Abstracts For Oral Presentations

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5 Drug Discovery Through Herbalomics James P. Tam School of Biological Science, anyang Technological University, Singapore. More than 80% of the world population, according to WH, depend on traditional medicines for primary care. Herbal medicine is the major form of traditional medicines, and has also provided important clues in the discovery of many smallmolecule drugs with MW <1 kda. At anyang Technological University, we have established a drug discovery and herbalomics program with the objective to expand beyond the conventional domain of natural products and chemical space of from small molecules of MW <1 kda to peptides and small proteins with MW 1-8 kda. Another objective is to discover, in this new domain and chemical space, novel bioactive peptides and small proteins of high metabolic stability and orally bioavailable as putative bioactive expected of herbal medicines. To achieve our objectives, our program aims to develop new platform technologies in proteomics, high put-through chemical synthesis and pharmacological assays in combination with geonomics and informatics. In this lecture. I will discuss a status report on our discovery and unpublished results on high put-through chemical synthesis.

6 James P. Tam Professor, Drug Discovery, School of Biological Sciences, anyang Technological University, Singapore James P. Tam is the Director of the Drug Discovery Laboratory. He served as the director of the double-degree program in Biomedical Science and Chinese Medicine, the Founding Dean of the School of Biological Sciences and the Founding Director of Biological Research Centre at anyang Technological University, Singapore. He received his Ph.D. in Medicinal Chemistry from the University of Wisconsin, Madison, USA and held appointments as Associate Professor at The Rockefeller University, USA ( ), Professor at Vanderbilt University, USA ( ) and The Scripps Research Institute, USA ( ). His research work focuses on the discovery, design and development of therapeutics, particularly orally active biologics, immunologics, anti-infectives, anti-proliferatives and synthetic vaccines. Professor Tam has published more than 330 papers in these areas of research. He received the Vincent du Vigneaud Award in 1986, the Rao Makineni Award by American Peptide Society in 2003, the Ralph F. Hirschmann Award by the American Chemical Society (ACS) in 2005, and the Merrifield Award by American Peptide Society in 2013 for his outstanding contributions to peptide and protein sciences. The Merrifield and Hirschmann Awards, administered by the APS and ACS respectively, recognize the highest achievements in the chemistry, biochemistry and biophysics of peptides at an international level. In addition to his scientific research, he has also been active in the peptide community. Besides serving on many editorial boards, he organizes international peptide and protein symposia and was co-founder of the past ten International Chinese Peptide Symposia. He received the Cathay Award from the Chinese Peptide Society, China in He was also honored as Honorary Professor by Peking University and Peking Union Medical College.

7 Peptide based materials: a bottom up approach G. Subra 1, S. Jebors 1 2, Soultan Al-Halifa 1, Cécile Echalier 1, Christine Enjalbal 1, Benjamin ottelet 1, Xavier Garric 1, Muriel Amblard 1, Ahmad Mehdi 2 and Jean Martinez 1 1 IBMM Institute of Biomolecules Max Mousseron Montpellier, UMR , Montpellier France 2 ICG Institute of Materials Charles Gerhardt, UMR 5253Montpellier France The preparation of hybrid materials containing peptides relies most of the time on physical entrapment of the peptide moiety or requires surface modification and ligation chemistries after the synthesis of the material 1. In contrast, we designed a new family of bioorganic-inorganic hybrid structured materials relying on the hybrid peptide-trialkoxsilyl building blocks that can be engaged directly in a sol gel process. This bottom up approach permits the introduction of any type of peptide in the material, providing different functionalities, biological and physicochemical properties. The hybrid peptide block can be either grafted or introduced by direct synthesis. Several examples will be presented including the synthesis of multifunctional nanoparticles for imaging, peptide-catalytic mesopous materials 2,3, anti fouling glass surfaces 4... At last, the self-assembling properties of the peptide unit can be exploited to yield a range of bio-inspired nanostructured materials. In an effort to develop polymerization systems suitable for peptide sequences, 5 we also describe a new class of silicon-peptides biopolymers. We used well defined chlorosilyl hybrid peptides as mononeric units, synthesized in solution or on dedicated solid support 6 to yield linear or comb-like peptide polymers. 1. G. Subra, A. Mehdi, C. Enjalbal et al., J Mater Chem 2011,.21 (17), S. Jebors, C. Enjalbal, M. Amblard et al., J Mater Chem B 2013, 1, Jebors, C. Enjalbal, M. Amblard et al., Tetrahedron 2013, 69, S. Jebors, C. Enjalbal, M. Amblard et al., J Mater Chem B 2013, 1, H. Enomoto, B. ottelet, S. Al Halifa et al., Chem Commun (4), Masurier, P. Zajdel, P. Verdie et al., Chem-Eur J (37),

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9 Gilles Subra graduated as a chemical engineer in 1994 from the Ecole ationale Supérieure de Chimie de Montpellier (France). In 1999, he received his Ph.D. from Montpellier University under the supervision of Professor Jean Martinez in the field of combinatorial synthesis of libraries of α-msh antagonists. He pursued his studies as an Institut Henri Beaufour postdoctoral fellow and set up the automated synthesis and analytical facility in faculty of Pharmacy as a tool for the discovery of new neuropeptides. In 2011, he obtained a Professor position at the University of Montpellier and is currently working in Max Mousseron Institute for Biomolecules (IBMM). His research topics, situated at the interface of chemistry, biology and analytical science, include combinatorial chemistry, supported synthesis, design of chemical tools for improving detection and quantification of biomolecules by mass spectrometry and peptide-based polymers and materials with a special interest in hybrid silicon containing bioorganic/inorganic materials.

10 Pro-survival Bcl-2 family proteins: structures, mechanisms and ligands Ho Sup Yoon Division of Structural Biology & Biochemistry School of Biological Sciences anyang Technological University Abstract Considering Bcl-2 family serving as a key homeostatic modulator between life and death pathways, targeting the pro-survival is certainly a promising anti-cancer therapeutic strategy. Extensive biochemical and structural studies provide insight into their canonical molecular mechanism of action and assisted designing molecules antagonizing protein-protein interaction between the pro-and anti-apoptotic Bcl-2 family proteins. n the other hand, alternative regulatory mechanisms of Bcl-2 family have been accumulating in recent years. FKBP38, a member of the immunosuppressive drug FK506-binding protein (FKBP) family, interacts with Bcl-2 and aids Bcl-2 s role in cell s survival pathways. MDM2-binding motif of p53 also interacts with various Bcl-2 family proteins in a manner similar to the modes of binding between the BH3 motifs of the pro-apoptotic Bcl-2 and ligand-binding pockets of the anti-apoptotic Bcl-2 family members, indicating that the tumor suppressor p53 and pro-apoptotic Bcl-2 family members may share a common molecular recognition mode with the anti-apoptotic Bcl-2 family. Taken together, versatile function of Bcl-2 family proteins certainly corroborate that they are involved in several cellular pathways and their versatile biological roles are well orchestrated in response to different stimuli perturbing cellular homeostasis. Structures, mechanisms, and inhibitors of the pro-survival Bcl-2 family proteins will be discussed in the talk.

11 Biography Ho Sup Yoon is currently the Head of Division, Division of Structural Biology & Biochemistry, School of Biological Sciences, anyang Technological University, Singapore. He received BSc degree from Seoul ational University, MSc degree from Korea Advanced Institute of Science and Technology (KAIST), and PhD degree from University of Chicago. He joined Abbott Laboratories in 1993 and contributed to various drug discovery projects and in particular played a pioneering role in deciphering biological function of Pleckstrin homology (PH) domain, identification of phosphatidylinositol 4,5-bisphosphate (PIP2) as PH domain s ligand, molecular mechanism of the pro-survival Bcl-2 family proteins and developing their inhibitors as anti-cancer drugs, which are currently in clinical trials. He left Abbott early 2002 and joined School of Biological Sciences, anyang Technological University as a founding faculty member. Prof. Yoon s current research programs focus on regulatory mechanism of Bcl-2 family proteins and roles of immunophilins in cancer, malaria, and neurodegenerative diseases.

12 Glycochemistry and Molecular Recognition Yannick Bessin, Véronique Barragan-Montero, Pascal Dumy, Alberto Marra, Jean-Louis Montero, Sébastien Ulrich, Jean-Yves Winum Institut des Biomolécules Max Mousseron (IBMM), Glycochemistry and Molecular Recognition team, ESCM, 8 Rue de l Ecole ormale, Montpellier Cedex 5, France [email protected] ur team has a recognized expertise in the development of chemoselective bioconjugation reactions as well as in the synthesis and study of template assembled multivalent systems using controlled, combinatorial and dynamic combinatorial chemistry. In particular, we have a large experience on the Cu-mediated azide-alkyne cycloaddition (affording 1,4-disubstituted triazole), the oxime bond formation (e.g. condensation of alkoxyamine with oxoaldehyde derivatives), and the thiol-ene and thiol-yne reactions (photoinduced radical addition of thiols to alkenes or alkynes) for the conjugation of carbohydrates with peptides, nucleic acids and other organic molecules. We have also demonstrated that some combinations of these click reactions are orthogonal and can be therefore used for regioselective functionalizations. Amongst the different research projects currently ongoing in our group, the following topics will be highlighted in the communication. Design and synthesis of zinc metalloenzymes activity modulators. This research program is dealing with the development of glyco-inhibitors of the carbonic anhydrase (CA) whose major enzymatic function is to catalyze the rapid reversible hydration of C 2 to bicarbonate and protons. In the context of tumors, one particular isoform, CAIX is associated with cancer progression, metastasis, and impaired therapeutic response. Carbohydrate-lectin interactions. ur research focuses on two lectins: the mannose-6-phosphate receptor (RM6P) and the rhamnose-binding lectin (RBL). In order to understand the role of these lectins, we are creating selectively functionalized multivalent platforms leading to non-covalent interactions. The synthesized nanoplatforms are nanoparticles as well as cyclodextrins and liposomes. Glycosidases inhibitors. While the multivalency approach has been extensively used by us and others in the carbohydrate-lectin recognition studies, much less is known in the field of glycosidase or glycosyltransferase inhibition. We have designed and synthesized new multivalent iminosugars exploiting calixarenes as platforms in order to evaluate the effect of valency and three-dimensional arrangements on the inhibition properties. Multivalent recognition of oligonucleotides. The interaction of artificial systems such as Peptide ucleic Acids (PA) scaffolds with oligonucleotides in a multi-point fashion is a promising avenue for enhancing binding affinity and selectivity. We have designed and synthesized modified amino acids featuring a bisfunctionalized non-natural base that can be used to prepare multivalent αpa. Dynamic materials for the recognition and transport of oligonucleotides. Dynamic materials that degrade into smaller components inside cells represent promising solutions for gene delivery. We have designed and synthesized acid-sensitive acyl-hydrazone-based dynamic polymers that combine cationic monomers and polyethylene oxide monomers, and we demonstrate that these materials effectively complex oligonucleotides.

13 Alberto Marra graduated in Pharmaceutical Sciences from the University of Pisa (Italy) in Then he moved to France and obtained his PhD in rganic Chemistry from the University Pierre et Marie Curie (Paris VI) in 1989 working in the laboratories of the Ecole ormale Supérieure under the supervision of Prof. Pierre Sinaÿ. He spent one more year in Paris as Researcher of the French ational Research Council (CRS). He was postdoctoral Research Associate at the University of Zurich (Switzerland) with Prof. Andrea Vasella (1991) and then moved to the University of Ferrara (Italy) where he joined the group of Prof. Alessandro Dondoni. He was appointed to a lectureship in rganic Chemistry at the Faculty of Engineering from 1992 to 1998 and was promoted to the position of Associate Professor in 1998 at the same University. Since 2002 he was teaching at the Faculty of Sciences of the University of Ferrara n September 2012 he left Italy to get a permanent position at the Université Montpellier 2 (France). His research interest centres on carbohydrate chemistry. In particular he synthesized oligosaccharide fragments of blood group determinants, heparin, dermatan sulfate and developed new glycosyl donors and glycosylation promoters. Then he developed new synthetic methodologies for the preparation of C-oligosaccharides, C-glycosyl amino acids, and C-glycosyl phosphonates. Recent work is dealing with the synthesis and molecular recognition properties of glycosylated crown ethers, glycoside clusters, glycodendrimers, monomeric and polymeric calixarene derivatives.

14 Potent health benefits from grape polyphenols and modified lipophenols Vercauteren J 1, Vigor C 1, Crauste C 1, Morel-Salmi C 2, Cazalet C 2, Durand T 1, Hamel C 4, Brabet P 4 1 Institute of Biomolecules Max Mousseron (IBMM), UMR 5247-CRS-UM1-UM2-ESCM, Faculty of Pharmacy, 15 av. C. Flahault Montpellier, France. 2 Caudalie Research Department, Laboratory of Pharmacognosy, Faculty of Pharmacy, 15 av. C. Flahault Montpellier, F. 3 Institute for eurosciences of Montpellier (IM), ISERM U1051-UM1 - UM2, Montpellier, F. [email protected] Phenols are powerful antioxidants, mostly as radical scavengers. This property relies first on the ability of the phenolic hydroxyle to ionize (low Bond Dissociation Enthalpy), thus facilitating the one electron delivery to the radical to be reduced, and second, on the aromaticity (hyperconjugation) of the newly formed aryloxy-radical which is then fully deactivated. Whatever the series: flavonoids (procyanidins, anthocyanins, ), or the minor stilbenoids (resveratrol monomers and oligomers; Figure 1), wine polyphenols are issued from the same mixed polyketide+skikimate precursor (the former through a Claisen cyclization by the chalcone-synthase, and the latter by the stilbene-synthase, via an aldol condensation). Thus, these two families of natural compounds are sharing, either readily or by a vinylogy principle, a 1,3,5-trihydroxybenzene (phloroglucinol-resveratrol) substructure, establishing an enhanced electronic density on the ortho and para carbon atoms (mesomeric effect, Figure 1). H H H aryloxy radicals (stabilized by conjugation) H R RH mesomeric effect BDE (Ar. /H. ) = 85 kcal/m radicalar reduction H H H "vinyls" H H phloroglucinol H flavanoids H stilbenoids (resveratrol) Figure 1. The two reducing and nucleophilic main properties of phenolic function. Therefore, not only such phloroglucinols are antioxidant, but also natural soft nucleophiles on those three positions, and thus efficient to scavenge electrophiles (carbonyl stressors), too. Because most of the pathologies (inflammation, cardiovascular and neurodegenerative diseases, cancer, ) are developing through these two cooccuring carbonyl and oxidative stresses (CS), there is a need for powerful and safe anti-cs active ingredients: this is the case of naturally occuring wine polyphenols. How this double anti-cs (carbonyl and oxidative stress) property is associated with beneficial health properties, will be presented. amely, which chemical modifications of the phenolic functions are able to adjust the softness/hardness nucleophilicity of these compounds (according to the Hard and Soft Acid and Base theory), to fight better against either ones of these pathologies, and aging, as well. H H H = nucleophilic centers

15 Pr VERCAUTERE Joseph, Louis Laboratory of Pharmacognosy, Faculty of Pharmacy UMR CRS 5247 IBMM University of Montpellier 1 15, Avenue Charles Flahault, MTPELLIER Cedex 5 [email protected] - Phone : University of REIMS Champagne-Ardenne: Scientific cursus Pharmacist: st PhD: Alkaloids of two Apocynaceae (director: Pr Jean LE ME, Reims) February 19 th, nd PhD: Total synthesis of vinblastine-type indole alkaloids (directors Pr Jean LÉVY and Dr Georges MASSIT) May 24 th, 1983 University of Wisconsin, MADIS, U.S.A: Post-Doctoral stay: chiral total synthesis with -allyl palladium complexes (Pr Barry M. TRST, Chemistry Department) Academic positions University of REIMS Champagne-Ardenne, Faculty of Pharmacy: Chargé de Recherche CRS: URA 492 (Pr Jean LE ME, Reims) University of BRDEAUX, Faculty of Pharmacy: Full Professor of Pharmacognosy: Study the properties, total synthesis, structural analysis of Polyphenols University of MTPELLIER, Faculty of Pharmacy: Full Professor of Pharmacognosy: 2002-present Industry connexion: Important discoveries allow us to develop polyphenolics anti-aging main applications in the cosmetic field (Caudalie ). Present position Professor of Pharmacognosy at the Faculty of Pharmacy, Department of SLB (Synthesis of Lipids Bioactive) IBMM - UMR CRS n 5247, University of Montpellier. Research Program: Development of reactive xygen Species (S) along with reactive Carbonyl Species (CS = sugars and lipids oxidation metabolites) scavengers (anti-cs), able to target retinal tissue and to slow down the pace of cytotoxic A2E formation (Stargardt disease). rganization of International Symposia President of 6 and Member of 12 scientific committees President of 7 and Member of 8 organizing committees Scientific production Patents and PCT: 37 International Publications: 112 International Invited Lectures: 83 Book Chapters: 92 ral Communications and Posters: 122 Supervision of PhD Theses: 18 Supervision of Masters: 17

16 Ghrelin Ligands: From Design and Synthesis to Clinical Studies. Toward a functional selectivity. Mathieu Maingot, Laurent Gavara, Céline M Kadmi, Séverine Denoyelle, Didier Gagne, Marjorie Damian, Sophie Mary, Jean Martinez, Jean-Louis Banères, Jacky Marie, Jean-Alain Fehrentz Institut des Biomolécules Max Mousseron (IBMM), CRS UMR 5247, Universités Montpellier 1 et 2, Faculté de Pharmacie, 15 avenue Charles Flahaut, 34093, Montpellier cedex 5, France. [email protected] The growth hormone secretagogue stimulating hormone receptor of type 1a (GHS- R1a) is activated by ghrelin, an acetylated 28-residue peptide hormone, to induce growth hormone secretion and food intake as well as to control energy homeostasis and reward seeking behaviors. We first developed a pseudo-peptide compound JMV 1843, orally active full agonist, which pasted the phase III clinical studies and will be soon commercialized for the GH deficiency diagnosis in adults. We then found an original series of ligands based on trisubstituted 1,2,4-triazoles. This family of compounds yielded to potent, selective agonists and antagonists, some of them exhibiting a selectivity of action. For example, compound JMV 2959 is able to totally inhibit food intake induced by an agonist, but is unable to inhibit GH secretion induced by the same agonist. This compound exhibited also anti-addictive properties against cocaine, amphetamines, alcohol

17 Jean-Louis Banères received a Ph.D. degree in Biochemistry from the University of Montpellier, in France. After a Ph.D. thesis work on the analysis of chromatin structure, he joined, in 1993, the Burnham Institute in La Jolla, California, as a postdoctoral fellow, to work on the molecular aspects of cell adhesion. He then joined the CRS, in France, in He is actually in the Molecular Pharmacology group at the Max Mousseron Institute for Biomolecules, in Montpellier (France). His research focuses on the analyses of the molecular bases of GPCR signaling using purified receptors. Jean-Alain Fehrentz received his PhD in chemistry from the University of ancy in 1983 and joined the Centre CRS-ISERM de Pharmacologie Endocrinologie of Montpellier, France in the Professor Bertrand Castro s group where his research was focused on the renin-angiotensin system. From 1989 to 1992, he was appointed as researcher in Sanofi Research and then moved to the School of Pharmacy of Montpellier under the direction of Professor Jean Martinez. He is currently holding a CRS Research Director position at the Institut des Biomolécules Max Mousseron (IBMM, UMR 5247). His research interests are the following: Synthesis of peptides, pseudo-peptides and peptidomimetics, Synthesis of exotic aminoacids from aminoaldehydes, Synthesis of peptide aldehydes, High-throughput chemistry, building blocks for combinatorial chemistry, new linkers, RCPG ligands (agonist or antagonist).

18 Preparation of ubiquitinated proteins using chemical ligation Liu Chuan Fa School of Biological Sciences, anyang Technological University, Singapore Abstract Ubiquitination is one of the most important protein posttranslational modifications in eukaryotic cells and regulates a great number of cellular processes, including protein degradation and gene expression. It refers to the linking of the C-terminus of ubiquitin protein (76 amino acids) to a particular lysine side chain of the target protein through an isopeptide bond. To study the functional roles of ubiquitination in mechanistic details, it is crucial to generate homogeneously ubiquitinated proteins. Biologically, ubiquitination is achieved through the consecutive action of three enzymes, ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2) and ubiquitin ligases (E3). Due to the difficulties in identifying or isolating the substrate specific ligases, enzymatic ubiquitination in vitro often has limited practical value. Chemical ubiquitination offers a better solution and has been the subject of intense research by several research labs. In the past several years, we have been engaged in developing methods for the synthesis of ubiquitinated peptides and proteins using chemical ligation. In this talk, I will present our earlier work on the -mercapto-lysinemediated ubiquitination as well as our more recent results on the use of a genetically incorporated unnatural amino acid for the site-specific ubiquitination of recombinant proteins.

19 LIU Chuan Fa Associate Professor School of Biological Sciences, anyang Technological University, Singapore Phone: (65) [email protected] EDUCATI PhD 1989 University of Montpellier (Faculty of Science), Montpellier, France BSc 1983 China Pharmaceutical University, anjing, China PRFESSIAL EXPERIECE present Associate Professor, School of Biological Sciences, anyang Technological University, Singapore Research Scientist IV Amgen Research, Amgen Inc., USA Research Scientist III Amgen Research, Amgen Inc., USA Research Scientist II Amgen Research, Amgen Inc., USA Research Scientist I Amgen Research, Amgen Inc., USA Research Instructor, Vanderbilt University Medical Centre, USA Postdoctoral Research Associate, Vanderbilt University Medical Centre, USA Postdoctoral Associate, The Rockefeller University, USA Research Associate, CRS, Centre CRS-ISERM de Pharmacologie-Endocrinilogie, Montpellier, France RESEARCH ITEREST Drug discovery: ew strategies for peptide drug development; Use of Fc-modified peptides (peptibodies) as therapeutic agents. Peptide chemistry and chemical biology: Development of chemical/enzymatic peptide ligation methods; Use of protein chemical synthesis for the study of protein structure and functions; Use of peptide nucleic acid analogs as antisense and antigene agents. MAJR RESEARCH ACCMPLISHMETS Co-inventor of the peptibody platform technology while working at Amgen, the world s largest biotech company. This work reveals that Fc-modified peptides have much better pharmacokinetic profiles than unmodified peptides and minimal immunogenicity. It represents a major advance in the field of peptide drug discovery, which has given Amgen several peptibody experimental drugs, including one already on the market. Work at Amgen was not published but was disclosed in patents/patent applications. Leading inventor of Amgen s first peptibody - plate (romiplostim), which was approved on August 22 nd by the FDA of USA for the treatment of adult chronic thrombocytopenia. Developed several peptide ligation methods (thiazolidine ligation, thioacid capture ligation, thioacid/azide amidation dual chemical ligation at lysine, etc.) for protein chemical synthesis and modification. Work in this area is of good international standing. AWARD anyang Award for Excellence in Teaching 2009 Technical and administrative staff (School core facility and double degree program): ur Eliza Binte sman, Li Fenxia, Qin Wei, Zhang Ying LIST F PATETS AD PUBLICATIS Patents (issued) 1. Liu, Chuan-Fa, Zeng Yun and Lu XiaoweiPeptide ucleic Acid Monomers and ligomers (2011). Singapore patent # (application filed in Sept 2008). 2. g, Gordon; Askew, Benny C., Jr.; D'Amico, Derin C.; Jarosinski, Mark A.; Liu, Chuan-Fa. Antagonists of the brandykinin b1 receptor (2009). US patent #7,605,120 (application filed in ct 2004). 3. Feige; Ulrich; Liu; Chuan-Fa; Cheetham; Janet C.; Boone; Thomas Charles; Gudas; Jean Marie (issued in 2009). Modified peptides as therapeutic agents. US Patent # 7,488,590 (application filed in Aug 2003) 4. P. Kostenuik P., C.-F. Liu, D.L. Lacey (issued in 2004). Modulators of receptor for parathyroid hormone and parathyroid hormone-related protein. US patent #6,756,480 (application filed in April 2001). 5. C.-F Liu, U. Feige, J. C. Cheetham. Thrombopoietic compounds (issued in 2004). US Patent #6,835,809 (application filed in ct 1999). 6. U. Feige and C.-F. Liu (issued in 2003). Modified peptides as therapeutic agents. US patent #6,660,843 (application filed in ct 1999). Selected recent publications 1 Jun-Feng Zhao, Xiao-Hong Zhang, Ying-Jie Ding, Yong-Sheng Yang, Xiao-Bao Bi, and Chuan-Fa Liu. 2 Facile Synthesis of Peptidyl Salicylaldehyde Esters and Its Use in Cyclic Peptide Synthesis. rg Lett. (2013), 15(20), Renliang Yang, Le Qi, Yanling Liu, Yingjie Ding, Milton Sheng Yi Kwek and Chuan-Fa Liu. Chemical Synthesis of -peptidyl Pyrrolidinemethanethiol for Peptide Ligation. Tetrahedron Letters (2013) 54 (29), R. Yang, W. Hou, X. Zhang, and C.-F. Liu. -to-c Sequential Ligation Using Peptidyl,-Bis(2-mercaptoethyl)amide Building Blocks. rg. Lett. (2012), 14, Y. Liu, C. Lu, Y. Yang, Y. Fan, R. Yang, C.-F. Liu,. Korolev, L. ordenskiöld. Influence of histone tails and H4 tail acetylations on nucleosomenucleosome interactions. J. Mol. Biol. (2011), 414, F. Li, A. Allahverdi, R. Yang, G. B. J. Lua, X. Zhang, Y. Cao,. Korolev, L. ordenskiöld and C.-F. Liu. A Direct Method for Site-specific Protein Acetylation. Angew. Chem. Intl. Ed. (2011), 50(41), C. H. Yi, H. Pan, J. Seebacher, I-H. Jang, S. G. Hyberts, G. J. Heffron, M. G. V. Heiden, R. Yang, F. Li, J. W. Locasale, H. Sharfi, B. Zhai, R. Rodriguez- Mias, H. Luithardt, L. C. Cantley, G. Q. Daley, J. M. Asara, S. P. Gygi, G. Wagner, C.-F. Liu and J. Yuan. Metabolic Regulation of Protein -Alpha- Acetylation by Bcl-xL Promotes Cell Survival. Cell (2011), 146 (4), Abdollah Allahverdi, Renliang Yang, ikolay Korolev, Yanping Fan, Curt A. Davey, Chuan-Fa Liu and Lars ordenskiold The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association. ucleic Acid Research (2011), 39, X. Zhang, F. Li and C.-F. Liu. Synthesis of histone H3 proteins by a thioacid capture ligation strategy. Chemical Communications (2011), 47(6), W. Hou, X. Zhang, F. Li and C.-F. Liu. Peptidyl.-Bis(2-mercaptoethy)-amides as thioester precursors for native chemical ligation. rganic Letters (2011), 13(3), Yang, K.K. Pasunooti, F. Li, X. Liu and C.-F. Liu. Synthesis of K48-linked diubiquitin using dual native chemical ligation at lysine. Chemical Communications (2010), 46, S. Thennarasu, Chuan-Fa Liu. A new safety-catch protecting group and linker for solid-phase synthesis. Tetrahedron Letters (2010), 51, K.K. Pasunooti, R. Yang, S. Vedachalam, B. K. Gorityala, C.-F. Liu and X. Liu Synthesis of 4-mercapto-L-lysine derivatives: potential building blocks for sequential native chemical ligation. Bioorganic & medicinal chemistry letters (2009), 19(22), R. Yang, K.K. Pasunooti, F. Li, X. Liu and C.-F. Liu. Dual native chemical ligation at Lysine. Journal of the American Chemical Society (2009), 131,

20 Post-polymerization modification of degradable polymers for biomedical applications Benjamin TTELET, Vincent DARCS, Hélène VA Den BERGHE, Xavier GARRIC, Jean CUDAE Institut des Biomolécules Max Mousseron - Département Biopolymères Artificiels (IBMM-BA) UMR CRS University Montpellier 1, University of Montpellier 2, ESCM Faculty of Pharmacy, 15 Av. C. Flahault, Montpellier, 34093, France [email protected] Although reported for more than 70 years, post-polymerization modification has gained tremendous attention in the last 10 years to yield functional polymers. In particular, the emergence of the click chemistry reactions in the field of macromolecular sciences allows the preparation of function-designed polymeric architectures. 1 In this presentation, we wish to report on the post-polymerization modification strategies used at the Artificial Biopolymers Department to provide functional biocompatible and biodegradable polymers for biomedical applications. Back in the early 2000s we developed an anionic activation methodology suited to the modification of FDA-approved aliphatic polyesters including the widely used PCL and PLA. 2 The key intermediate of this strategy is a macropolycarbanion that can react either with electrophiles to yield substituted polyesters, or as an anionic macroinitiator to yield graft copolyesters. 3,4 The versatility of this approach relies on the possibility to apply it in solution or for surface reactions, as well as in the possibility to combine it with other macromolecular tools like photochemistry, click chemistry and controlled-radical polymerization. Various recent achievements of the group in relation with hot biomedical applications including MRIvisible polyesters for diagnostic, antibacterial surfaces will be presented Theato P. and Klok H-A. Eds. Functional Polymers by Post-polymerization Modification, Wiley-VCH,Weinheim, Germany Ponsart S. et al. A novel route to poly(epsilon-caprolactone)-based copolymers via anionic derivatization. Biomacromolecules 2001, 1, ottelet B. et al. Synthesis of an X-ray opaque biodegradable copolyester by chemical modification of poly (epsiloncaprolactone). Biomaterials 2006, 27, ottelet B. et al. ovel amphiphilic poly(epsilon-caprolactone)-g-poly(l-lysine) degradable copolymers. Biomacromolecules 2007, 8, Blanquer S. et al. ew magnetic-resonance-imaging-visible poly(epsilon-caprolactone)-based polyester for biomedical applications. Acta Biomater. 2012, 8, El Habnouni S. et al. Mild Methodology for the Versatile Chemical Modification of Polylactide Surfaces: riginal Combination of Anionic and Click Chemistry for Biomedical Applications. Adv. Funct. Mater. 2011, 21, El Habnouni S. et al. Toward potent antibiofilm degradable medical devices: A generic method for the antibacterial surface modification of polylactide. Acta Biomater. 2013, 9, 7709.

21 Benjamin ottelet é le 15 janvier 1978 à Paris 14ème (75) Maître de Conférences HDR (section 85) à la Faculté de Pharmacie, Université Montpellier 1 avec rattachement de recherche au sein du Département Biopolymères Artificiels de l Institut des Biomolécules Max Mousseron. Thématiques: Synthèse, modification et mise en forme de nouveaux polymères biodégradables pour applications biomédicales et pharmaceutiques. Parcours : : Maître de conférences à la Faculté de Pharmacie, Université Montpellier : Post-doctorat à l Université de Genève, groupe FAGAL (Pr. Guny) : ATER ESCM, groupe IAM (Pr. Boutevin) : Doctorat, Université Montpellier 2, groupe CRBA (Pr. Vert) : Ingéniorat ESCM Données bibliométriques Auteur et co-auteur de 27 publications et 1 brevet Auteur et co-auteur de 27 communications orales internationales et 21 communications par affiche Résumé d'encadrement Thèses: 3, Doctorants étrangers dans le cadre de collaborations: 2, Post-doctorants: 3 Master 2: 6, Master 1 et Licence 3 > 20 Prix et récompenses 2008 : European Society Artifical rgans : Wichtig Award 2014 : Prix Chercheur d Avenir LR projet VIP-Biomat Responsabilités administratives et de recherche : Responsable d'ue "Polymères d'intérêt thérapeutique" L3 Ingénierie de la Santé : Membre du Conseil de l'ecole Doctorale Sciences Chimiques Balard (ED 459) : Membre du bureau de la Commission Recherche de l UM : Expert auprès de l'bservatoire des Micro et anotechnologies (MT) : Membre élu au Conseil d UFR (collège B)

22 Insight into the Programming Mechanism of Iterative Polyketide Synthases Zhao-- Xun Liang School of Biological Sciences, anyang Technological University Type I iterative polyketide synthases (PKS) are multifunctional enzymes that assemble polyketide products from acetate units by using a single module of catalytic domains. The use of a single module of catalytic domains makes the iterative PKSs mechanistically distinct from the type I modular PKSs that employ multiple modules of catalytic domains. The structural diversity exhibited by the products of partially reducing PKSs arises from the programming of the catalytic domains, through the control of the carbon chain length of the polyketide intermediates and the keto-- reduction pattern. Elucidation of the molecular determinants of the carbon chain length and keto-- reduction pattern is crucial for deciphering the programming mechanism of partially reducing PKSs. We recently discovered a novel partially reducing PKS that exhibits a distinct keto-- reduction pattern. By comparative studies, we established that the ketoreductase domain of the partially reducing PKS is solely responsible for the programmed ketoreduction. We identif ie d a key residue in the ketosynthase domain for controlling the carbon-chain length of the linear polyketide. We found that the programming of the partially reducing PKSs can be altered by rational grafting of the active-- site motifs of the ketoreductase domain. The results together shed light on the programming mechanism and demonstrate the potential of redesigning these PKSs for the production of natural product derivatives.

23 Zhao-Xun Liang studied rganic Chemistry at Lanzhou University in China, where he received B.S and M.S. degrees under the guidance of Prof. Qing-Xiang Guo. He obtained his Ph.D. degree in Chemistry from orthwestern University under the supervision of Brian Hoffman ( ). He worked with Judith Klinman for three years as a postdoctoral fellow at the University of California at Berkeley. In 2005, he joined the School of Biological Sciences of anyang Technological University (TU) in Singapore as an assistant professor. He is currently an associated professor with interest in bacterial biofilm and secondary metabolite biosynthesis.

24 Solid-Phase Chemical Synthesis of 5 -triphosphate RA, 5 -capped RA and their analogs with modified triphosphate chain, substrates of viral enzymes Thillier Y 1, Sallamand C 1, Decroly E 2, Canard B 2, Vasseur J-J 1, Debart F 1 1 IBMM, UMR 5247 CRS-UM1-UM2, DACA, Université Montpellier 2, Place Eugène Bataillon, Montpellier, France; 2 AFMB, UMR 7257 CRS-Université Aix-Marseille, 163 Avenue de Luminy, Parc Scientifique et Technologique de Luminy, Marseille cedex 09, France [email protected] Eukaryotic cellular messenger RA (mra) and many viral mra bear a 5 -cap structure ( 7m Gppp) which consists of a 7 -methylguanosine linked to the first nucleotide () of premra transcript by a 5-5 -triphosphate bridge. This cap moiety is a critical structure involved in various cellular processes of gene expression. Most of RA viruses developed their own enzymatic capping pathway to enhance both their genome stability and translation. Thus, 5 -triphosphate (TP) RA and 5 -capped RA are high valuable molecules which serve as important substrates for structural and mechanistic studies of biological processes such as protein translation, mra turnover, or to identify novel enzymatic targets for antiviral drug design. To date, they are produced by enzymatic processes in low yields with a weak variability of the 5 -end. To overcome this bottleneck we aimed to develop a chemical access on solid-support to these 5 -functionalized RA in great amount without any limitations on RA sequence. Here, a robust, reproducible, and scalable method for the solid-phase synthesis of 5 - triphosphate RA will be presented 1,2. Furthermore, this strategy was extended to produce enzymatic resistant analogs of the triphosphate counterpart like: 5 -(β,γ-methylene)-tp, 5 - (α-p-thio)-tp et 5 -(α-p-thio)-(β,γ-methylene)-tp RA 3. A similar approach was developed for the production of 5 -capped RA following a two-steps process. First the coupling of the cap structure was performed on solid-supported RA then an enzymatic 7 -methylation was achieved in solution 4-6. Using the same route we succeeded as well in the synthesis of 5 -capped analogs RA bearing a 5 -β,γ-methylene bridge or a 5 -(α-p-thio)-modification within the triphosphate moiety. 1. Lavergne, T.; Bertrand, J.-R.; Vasseur, J.-J.; Debart, F. Chem. Eur. J. 2008, 14, Zlatev, I.; Lavergne, T.; Debart, F.; Vasseur, J.-J.; Manoharan, M.; Morvan, F. rg. Lett. 2010, 12, Piton, J.; Larue, V.; Thillier, Y.; Dorléans, A.; Pellegrini,.; Sierra-Gallay, I. L. d. l.; Vasseur, J.-J.; Debart, F.; Tisné, C.; Condon, C. Proc. atl. Acad. Sci. U.S.A. 2013, 110, Thillier, Y.; Decroly, E.; Morvan, F.; Canard, B.; Vasseur, J. J.; Debart, F. RA 2012, 18, Selisko, B.; Potisopon, S.; Agred, R.; Priet, S.; Varlet, I.; Thillier, Y.; Sallamand, C.; Debart, F.; Vasseur, J.-J.; Canard, B. PLoS Path. 2012, 8, e Barral, K.; Sallamand, C.; Petzold, C.; Coutard, B.; Collet, A.; Thillier, Y.; Zimmermann, J.; Vasseur, J.-J.; Canarda, B.; Rohayem, J.; Debart, F.; Decroly, E. Antiviral. Res. 2013, 99,

25 Dr. Françoise DEBART Françoise Debart received her PhD in rganic Chemistry (1989) at University of Montpellier under the supervision of Prof. Imbach and Dr. Rayner on the molecular recognition of nucleic acids by pseudopeptides or oligonucleotides for gene expression regulation. In 1989 she obtained a permanent position at CRS in Montpellier working on modified oligonucleotides for antisense applications. From 1990 to 1992 she was a visiting scientist at Isis Pharmaceuticals in California (USA), company expert in the antisense field. She then came back to University of Montpellier to develop new modified oligonucleotides for antisense applications. Since 2011 she has been a director of research at CRS. She is currently managing projects on various aspects of RA chemistry in oligonucleotides team directed by Dr. Vasseur. Her research interests focus on RA modifications for applications in RA interference field, and RA maturation and processing. f particular interest, she developed new methods for chemical synthesis of regular RA, 5 -triphosphate RA and 5 -capped RA and their triphosphate analogues.

26 Advances in the development of nucleotide analogues targeting 5 -nucleotidase c-ii Peyrottes S. 1, Jordheim; L. P. 2, Lionne C. 3, Chaloin L. 3, Dumontet C. 2 and Périgaud C. 1 1 IBMM, UMR 5247-CRS-UM 1 & 2, Montpellier, France; 2 CRCL, ISERM U590, Lyon, France; 3 CPBS, UMR 5236-CRS-UM 1 & 2, Montpellier, France. [email protected] 5 -ucleotidases belong to the haloacid dehalogenase (HAD) family and regulate the intracellular nucleotide pools by catalyzing the dephosphorylation of nucleoside 5 -monophosphates to the corresponding nucleosides and inorganic phosphate. 1 These eight enzymes differ in substrate specificity, sub cellular location and tissuespecific expression. Thus, intracellular 5 -nucleotidases are likely to affect the phosphorylation level and the pharmacological activity of nucleoside analogues used in the treatment of cancers and viral diseases. Cytosolic 5 -nucleotidase II (c-ii) plays a pivotal role in purine cellular metabolism and therefore appears of potential therapeutic importance. Indeed, in the literature few studies have dealt with the possible role of c-ii in drug resistance mechanism and/or in cell death by itself. 2 During the last few years, we have developed a pluri-disciplinary program to identify and to characterize potential inhibitors of c-ii. In this respect, series of nucleosidic phosphonate analogues (incorporating a chemically and enzymatically stable P-C bond) were designed as mimics of nucleoside 5 -monophosphates, the natural substrate of c-ii. ver the course of our study, a β-hydroxyphosphonate derivative has been identified as a lead-compound. In silico docking, synthesis, biological evaluation and results from our structureactivity relationship (SAR) studies will be presented in details. 3 This study gives new structural insights for the development of both structure-based and pharmacophorebased inhibitors. [1] Walldén K. et al. J. Biol. Chem. 2007, 282, (b) Hunsucker S.A. et al. Pharmacol. Ther. 2005, 107, [2] Galmarini C. et al. Br. J. Haematol., 2003, 122, 53-60; Careddu, M. G. et al. Biochim. Biophys. Acta, 2008, 1783, ; L.P. Jordheim & L. Chaloin, Curr. Med. Chem., 2013, 20, ; J. Zavadil, et al. at. Genet., 2013, 45, ; G. Tzoneva, et al. at. Med., 2013, 19, [3] F. Gallier, et al. PLoS Computational Biology, 2011, 7, e ; M. Meurillon, et al. Eur. J. Med. Chem. 2014, in press.

27 Suzanne Peyrottes, PhD, 44 years old, French Senior Scientist (DR2) at CRS Institut des Biomolécules Max Mousseron (IBMM), UMR5247-CRS UM1 UM2, University Montpellier 2, Place E. Bataillon, Montpellier, France. Tel: +33 (0) ; Fax: +33 (0) EDUCATI - Diplôme d'etudes Approfondies (D.E.A.) obtained with Honors, June 2003 Chemistry of Biomolecules: Synthesis, Structure and Reactivity - PhD : ovember 1995, Speciality: rganic, Mineral, Analytic and Industrial Chemistry Research carried out in the Laboratory of Bioorganic Chemistry (Director: Prof. J.-L. Imbach), University of Montpellier II. Topics developed: - Synthesis and study of new oligonucleotides and dinucleosidic entities with neutral internucleosidic backbones (phosphotriester, phosphoramidates). - ligonucleotides incorporating two kinds of modifications such as the anomery of the nucleoside and the internucleosidic backbone. - Habilitation à Diriger des Recherches (H.D.R.), June 2003 PRFESSIEL APPITMETS April December 1997 : Post-doctoral position in the laboratory of Dr M.J. Gait, Medical Research Council (MRC), PAC division, Cambridge, UK. Research Topics: Solid-phase synthesis of oligonucleotide - peptide conjugates in order to improve their intracellular targeting. March September 1999 : Post-doctoral position in the laboratory of Prof. J.-L. Imbach, UMR 5625 CRS-UM II, Université Montpellier II, France. Research Topics: Design, synthesis and study of antitumoral mononucleotide prodrugs. ctober : Senior Scientist (CRS CR1). TPICS of ITEREST - Targeting of phosphorylated effectors. - Synergetic effect in antiviral chemotherapy: Design of bifonctional phosphorylated entities. - Synthesis and study of phosphonate analogues as potential therapeutic agents - Development of choline analogues as anti-malarial agents SCIETIFIC WRK 51 publications in peer-reviewed journals, 5 patents, 3 book chapters 34 Invited presentations and ral communications 55 Posters VARIUS RESPSABILITIES Member of the organization committee of the «IV th Cambridge Symposium for ligonucleotide Chemistry and Biological Applications», September 2-5, 1997, Cambridge, UK. Member of the scientific and organization committee of the «XIII th International Round Table: ucleosides, ucleotides and Their Biological Applications», September 6-10, 1998, Montpellier, France. Member of the scientific and organization committee of the II nd Symposium of Montpellier Infectious Diseases», ovember 29-30, 2012, Montpellier, France. Member of the International Society for ucleosides, ucleotides and ucleic Acids (IS3A) since 1995, Member of the IS3A membership committee, and the International Society for Antiviral Research.

28 Dissecting the dengue virus replication complex for the discovery of antiviral inhibitors. Julien Lescar Division of Structural Biology and Biochemistry School of Biological Sciences anyang Technological University Dengue is a major public-health problem: hundreds of thousands cases of the severe hemorrhagic form of the disease occur every year and a large and increasing proportion of the world population is at risk. Recent phase IIb clinical trials in Thailand revealed that the Sanofi tetravalent vaccine failed to confer cross-protection against one of the four Dengue virus (DEV) serotypes. Specific drugs have yet to be approved to treat dengue or other conditions caused by related flaviviruses, such as West-ile virus and Japanese encephalitis virus. In the absence of a vaccine conferring true and lasting cross-protection against the four -and possibly fiveserotypes of DEV, outbreak control and patient care has to rely on symptomatic treatment and specific antiviral molecules. Plus-strand RA virus replication occurs in association with cytoplasmic host-cell membranes, where both viral and cellular host factors cooperate within an organelle-like replication factory called replication complex (RC). Several non-structural proteins of the RC constitute validated drug targets because of their crucial functions during viral replication. However, a major impediment in developing drugs targeting the dengue virus (DEV) RC is that both its morphology and composition, the interplay between its molecular constituents as well as the precise molecular mechanisms for viral RA replication are still elusive. ver the last decade, individual protein components of the RC were characterized both at the functional and structural level. However, a main challenge lying ahead is to gain a better understanding of the entire flavivirus RC. We will summarize our current knowledge of the flavivirus RC and present our recent studies on S4B and S3 revealing protein-protein and protein-ra interactions within the DEV RC.

29 Julien Lescar, Associate Professor anyang Technological University A. Academic Qualifications : Ph. D. in Biological Crystallography : Master Degree in Physics University of PARIS XI, rsay B. Professional experience Since January 2002 Associate Professor (January 2004, with tenure: 2008) with the School of Biological Sciences, anyang Technological University of Singapore Scientific Collaborator ovartis Institute for Tropical Diseases Adjunct Associate Professor Duke US Graduate Medical School (2008). Since ctober 1999: Permanent Staff scientist at the C..R.S. Scientific Collaborator at the ESRF (European Synchrotron Radiation Facility) Grenoble, France : Staff Scientist with the ESRF-EMBL Joint Structural Biology Group Co-responsible for the High-Brillance Beamline ID2 for Biocrystallography, Grenoble, France : Post-doctoral fellow Institut Pasteur, Paris, France 1994: Post-doctoral fellow, Washington University School of Medicine, St Louis, : Graduate Student, Institut Pasteur, Paris, France. Research interests : X-ray crystallography, Structure of RA viruses, Structure-based drug discovery. Scientific awards Fellowships from Ministère de la Recherche Fellowship from Pasteur-Weiszmann in 1993 Scientific Collaborator ovartis Institute for Tropical Diseases Scientific Collaborator at the European Synchrotron Radiation Facility Relevant publications: 1- Zou J, Xie X, Lee LT, Chandrasekaran R, Reynaud A, Yap L, Wang Q-Y, Dong HP, Kang C, Yuan Z, Lescar J* & Shi P-Y (2014) Dimerization of flavivirus S4B protein J. Virol. Jan 3 2- Xie X, Zou J, Wang Q-Y, oble CG, Lescar J, Shi P-Y (2014) Generation and characterization of two mouse monoclonal antibodies against S4B protein of dengue virus. Virology , Lim SP, Hong J, Seh CC, Liew CW, Davidson A.D., Chua LS, Chandrasekaran R, Cornvik T, Shi P-Y & Lescar J (2013) A crystal structure of the Dengue virus S5 polymerase delineates inter-domain amino acids residues that enhance its thermostability and de novo initiation activities. J. Biol. Chem. 288, Decroly E, Ferron F, Lescar J*, Canard B (2012) Conventional and unconventional mechanisms for capping of virus mra. ature Reviews Microbiology 10, Zou G, Chen Y-L, Dong H, Lim CC, Yap LJ, Yau YH, Lescar J, Shi P-Y (2011) Functional analysis of two cavities in flavivirus S5 polymerase. J. Biol. Chem. 286, Ferron F, Li Z, Danek EI, Luo D, Wong YH, Coutard B, Lantez V, Charrel R, Canard B, Walz T & Lescar J*(2011) The hexamer structure of the Rift Valley Fever virus nucleoprotein suggests a mechanism for its assembly into ribonucleoprotein complexes. PLoS Pathogens /journal.ppat Decroly E, Debarnot C, Ferron F, Bouvet M, Coutard B, Imbert I, Gluais L, Papageorgiou, rtiz-lombardia M, Lescar J & Canard B (2011) Crystal structure and functional analysis of the SARS-Coronavirus RA cap 2 Methyltransferase nsp10/nsp16 complex. PLoS Pathogens May;7(5):e Lim SP, Sonntag LS, oble C, ilar SH, g RH, Zou G, Monaghan P, Chung KY, Dong H, Liu B, Bodenreider C, Lee G, Ding M, Chan WL, Wang G, Yap LJ, Chao AT, Lescar J, Yin Z, Vedananda TR, Keller TH, Shi P-Y (2011) Small-molecule inhibitors that selectively block dengue virus methyltransferase. J. Biol. Chem. 286, Liew CW, Sharff A., Kotaka M., Kong R., Sun H., Qureshi I.A., Bricogne G., Liang ZX & Lescar J.* (2010) Inducedfit upon ligand binding revealed by crystal structures of the hot-dog fold thioesterase in Dynemicin biosynthesis. J. Mol. Biol. 404: Luo D, Wei, Doan D, Paradkar P, Chong Y, Davidson A, Kotaka M, Lescar J*, Vasudevan SG (2010) Flexibility between the protease and helicase domains of the dengue virus S3 protein conferred by the linker region and its functional implications. J. Biol. Chem. 285, Lescar J* & Canard B (2009) RA-dependent RA polymerases from flaviviruses and picornaviridae. Curr pin in Struct. Biol. 19, Luo D, Xu T, Watson RP, Scherer-Becker D, Sampath A, Jahnke W, Yeong SS, Wang CH, Lim SP, Strongin A, Vasudevan SG & Lescar J.* (2008) Insights into RA unwinding and ATP hydrolysis by the flavivirus S3 protein. EMB J. 27(23):

30 Taylor dispersion analysis for the characterization of nanomaterials Hervé Cottet 1, Jean Philippe Biron 1, Joseph Chamieh 1, Laurent Leclercq 1, Michel Martin 2, Luca Cipelletti 3 1 Institut des Biomolécules Max Mousseron (UMR 5247 CRS - Université de Montpellier 1 - Université de Montpellier 2), place Eugène Bataillon CC 1706, Montpellier Cedex 5, France. 2 Ecole Supérieure de Physique et de Chimie Industrielles, Laboratoire de Physique et Mécanique des Milieux Hétérogènes (PMMH, UMR 7636 CRS, ESPCI-ParisTech, Université Pierre et Marie Curie, Université Paris-Diderot), 10 rue Vauquelin, Paris Cedex 05, France 3 Laboratoire Charles Coulomb, UMR CRS 5221, Université de Montpellier 2, place Eugène Bataillon, Montpellier Cedex 5, France. [email protected] Taylor Dispersion Analysis (TDA) is an absolute and straightforward method for the determination of molecular diffusion coefficients (D), and thus, hydrodynamic radii (R h ). TDA can be used for sizing solutes of different natures (nanomaterials or nanoparticles, proteins, (co)polymers, dendrimers, liposomes, small molecules ) and of virtually any size from angstrom to sub-microns. The main advantages of this method are: low sample consumption (nl injected), fast and simple analysis without calibration, determination of weight-average R h value for mass concentration sensitive detection, low sensitivity to dusts compared to dynamic light scattering techniques, application to the size-characterization of complex mixtures (monitoring of polymerization processes, polymer degradation, aging in solution, self assembly ) and inline coupling to capillary electrophoresis. In this presentation, the basics of TDA will be presented. Examples of applications for the characterization of mono- and polydisperse samples (dendrigraft poly-l-lysine, proteins, synthetic polymers, nanoparticles, polymeric drug delivery systems) will be presented including comparison with dynamic light scattering experiments. It will be demonstrated that the deconvolution of the taylorgrams can be performed to get the hydrodynamic radius distribution (and not only the average R h ).

31 CV-Biography Hervé Cottet completed his PhD in analytical chemistry in 1999 at the Ecole ationale Supérieure de Chimie de Paris (ESCP, ParisTech, France) under the supervision of Prof. Pierre Gareil. He worked on Capillary Electrophoresis (CE) of synthetic polyelectrolytes. After a one year post-doc at the Technical University of Eindhoven (the etherlands), where he investigated the use of non aqueous solvents in CE, he joined the University of Montpellier in 2000 as an assistant professor. In 2007, he obtained a full professor position at the Max Mousseron Biomolecules Institute in Montpellier. His research work concentrates at the interface between separation sciences, polymers and biology. He is developing CE methodologies (2D-separations, free solution and gel-based separations, micellar / microemulsion modes, isotachophoresis, isoelectric focusing) and Taylor Dispersion Analysis (TDA) for the characterization of (bio)polymers, polyelectrolytes, dendrimers, nanoparticles, colloids and bacteria. He is both interested in fundamentals (mobility modeling, electrophoretic behavior) and practical (or industrial) applications of CE and TDA. In 2006, he awarded the price from the Analytical Chemistry Division of the French Chemical Society. In 2011, he has been nominated as a junior member of the Institut Universitaire de France. He is a member of the directorial board of the french-speaking separation science society (AfSep).

32 Exploring frontiers of peptides synthesis through mechanochemistry Julien Bonnamour, Thomas-Xavier Métro, Jean Martinez and Frédéric Lamaty Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CRS-Universités Montpellier 1 et 2, Université Montpellier 2, Place Eugène Bataillon, Montpellier cedex 05, France. [email protected] Classical amides and peptides synthetic methods suffer from various limitations: - large amounts of reagents and solvents are needed - required solvents are often very toxic, - production of large quantities of waste, - low productivity, - side reactions due to highly diluted reaction media, - peptide size limitations due to aggregation We have recently shown that using mechanochemistry can help in solving these limitations. 1, 2 The use of high-energy ball-milling has allowed us to produce various peptides by treating stoichiometric amounts of activated amino acids (Boc-AA-Su and UCA) with amino acid alkyl ester salts in the presence of innocuous ahc 3 and EtAc. Di- to penta-peptides such as Leu-enkephalin were produced in high yields without requiring tedious chromatography purifications (Scheme 1). 2 Efficient synthesis of Boc-(Leu) 4 -Bn, which is highly prone to aggregation, let us envisage excellent abilities for this mechanically-assisted approach to enable the synthesis of other difficult and much longer peptide sequences. Mechanically-assisted peptide synthesis 1. Declerck, V.; un, P.; Martinez, J.; Lamaty, F., Solvent-Free Synthesis of Peptides. Angew. Chem. Int. Ed. 2009, 48, Bonnamour, J.; Métro, T.-X.; Martinez, J.; Lamaty, F., Environmentally benign peptide synthesis using liquid-assisted ball-milling: application to the synthesis of Leu-enkephalin. Green Chem. 2013, 15,

33 CV Thomas-Xavier Métro After having spent one year in USA at Atofina Chemicals working on organic peroxides safety process research, I graduated with a Master s degree in general chemistry from the Ecole Supérieure de Chimie rganique et Minérale and a Master's degree in medicinal biochemistry from the Université de Pharmacie de Montpellier both in I achieved my PhD in organic chemistry in 2008 from the Université Pierre et Marie Curie (Paris) under the supervision of Dr Domingo Gomez Pardo and Professor Janine Cossy. My research work was dedicated to the rearrangement of amino alcohols and its application to the synthesis of (S,S)- Reboxetine and SSR After a first post-doc in process R&D at Sanofi Chimie in Sisteron and second post-doc in medicinal chemistry at Sanofi-Aventis R&D in Montpellier, I joined the CRS in 2011 as a Chargé de Recherche in the Green Chemistry and Enabling Technologies Team. Since then my research activity is based on the development of solvent-free methodologies, aiming to provide the organic chemist a set of solutions to reduce the environmental impact of his chemical enterprise.

34 Development of a humanized mouse model for the development of new therapies against malaria Peter R. Preiser Molecular Genetics & Cell Biology School of Biological Sciences anyang Technological University Abstract Malaria remains a serious global health problem with million clinical cases being reported each year. Successful control and eradication of this infectious disease will require a combination of vector control, drug therapy and ideally an effective vaccine. To date no effective vaccine is available and there is a growing concern about the spread of insecticide resistance and drug resistance against all approved drugs by the parasite. While animal models have provided powerful insights on the biology of the malaria parasite they have been extremely poor substitutes for the development of an effective vaccine targeting the human forms of this parasite. The recent evaluation of a human vaccine RTS/S targeting Plasmodium falciparum the parasite that causes the most severe disease in humans has been hugely expensive and unfortunately the overall results are disappointing. There is therefore an urgent need to develop an effective animal model that efficiently emulates the human immune response and allows the direct evaluation of human vaccine candidates. To achieve this goal we have developed a humanized mouse model that has been transplanted with an efficient human immune system. At the same time this mouse is able to support the efficient replication of P. falciparum and therefore represents an ideal system to test new therapeutic approaches against the parasite. Using this model we have identified a number of key immune effector cells that are important in controlling parasite replication. In addition we have for the first time tested a therapeutic antibody in an in vivo system. These results clearly indicate that the humanized mouse system is a viable early test model for P. falciparum therapeutics.

35 Biography Peter Preiser is a professor of Molecular Genetics & Cell Biology and Chair of the School of Biological Sciences at the anyang Technological University (TU). He obtained his PhD in Biology from the University of Delaware, USA, in After his postdoctoral appointment at Worcester Foundation for Experimental Biology, USA, he joined London s ational Institute for Medical Research as a Senior Research Scientist. In 2003 he left London to join TU s School of Biological Sciences (SBS). He has extensive experience in malaria biology and developing quantitative proteomic approaches along with transcriptional profiling to understand complex biological processes in relation to host parasite interaction. He is also an active member of the infectious disease inter-disciplinary research group with the Singapore-MIT Alliance for Research and Technology (SMART) and has collaborated with top research institutes around the world and has published over 60 top-quality international journal papers.

36 Study of both large and small amplitude motions of G-Proteins Coupled Receptors (GPCRs) at the atomic scale by different molecular modelling approaches. icolas FLQUET a, Maxime LUET a, Landry CHARLIER a, David PERAHIA b, Jean MARTIEZ a a Institut des Biomolécules Max Mousseron (IBMM, CRS UMR5247), Faculté de Pharmacie, 15 avenue Charles Flahault, BP 14491, Montpellier Cedex 05, France b Laboratoire de Biologie et Pharmacologie Appliquée (LBPA), CRS UMR8113, École ormale Supérieure de Cachan, 61 Avenue du Président Wilson, Cachan Cedex, France *[email protected] G-Proteins Coupled Receptors form a large family of trans-membrane proteins and are the target of ~40 % of actual marketting drugs. Despite the numerous X-ray structures published these last years in the field, many questions still remain unsolved concerning the activation of these large molecular systems. From Biochemical, Biophysical and Structural data, we indeed know that the multi-step activation of GPCRs and protein partners at the atomic scale is a dynamical process that both require small and large amplitude motions. Starting from existing X-ray structures in the PDB, we have combined different molecular modelling approaches including Molecular Dynamics Simulations and ormal Modes Analyses (MA) to identify the motions putatively involved in extracellular ligand binding, GDP release, dissociation of the G-proteins at proximity of the membrane, etc... Among other results, we recently observed that the receptor was able to adopt only one major motion in the GPCR:G-protein complex. This motion corresponded to an anti-symmetric rotation of both its extra- and intra-cellular parts, with a key role of previously identified highly conserved proline residues. Because this motion was also retrieved when performing MA on 7 other GPCRs which structures were available, it is strongly suspected to possess a significant biological role, possibly being the "activation mode" of a GPCR when coupled to G-proteins. Discussion will be made on how such a motion could be favored by the presence or not of a ligand, and how agonists, antagonists and inverse agonists could act differently on these very important proteins

37 Dr. icolas FLQUET CRS Research Assistant (CR1, section 16), HDR Born in Reims (France) (37 years old) Married, 2 children [email protected] Lab.: (+33)(0) ; Fax: (+33)(0) Port.: (+33)(0) EDUCATI / RESEARCH EXPERIECE Juin 2011 Habilitation à Diriger les Recherches (HDR) University Montpellier 1, Montpellier. ct CRS Research Assistant IBMM CRS UMR5247, Montpellier Post-doctorate Fellow ICS CRS UPR2301, Gif sur Yvette «Attaché Temporaire d Enseignement et de Recherches» Université de Reims Champagne Ardenne, Reims PhD Thesis in Molecular Modelling University Paris 7 Denis Diderot, Paris. Since 2007 : Supervision of the Molecular Modelling group of IBMM Supervision of one Post-Doc Landry CHARLIER ( ) Coarse Grained Models applied to GPCRs ( ). Supervision of the PhD thesis of Maxime LUET ( ) Study at the molecular level of G-proteins and their coupled Receptors. Supervision of two master degree students (2011 & 2012): Esra KARAKAS and Alexandre PERRET on Large collective motions of GPCRs predicted from their X-ray structures. Regular «reviewer» in the following journals: Arch.Biochem.Biophys., Biochemistry, Journal of Molecular Modelling, Journal of rganic Chemistry, JACS, etc... Expert for the French ational research agency (AR) and the Swiss ational Science Foundation (SSF) and Expert for PRACE (Partnership for Advanced Computing in Europe) Member of the french Peptide & Proteins Group (GFPP, Member of the ational Research Group (GDR-3545) GPCR PhysioMed ( and of the Management Committee of the CST action «GLISTE»: GPCR-Ligand Interactions, Structures, and Trans-membrane : a European etwork. PRIZES and AWARDS 2012 Bronze Medal of CRS, Chemistry ational Institute (IC), section 16 MST RECET REPRESETATIVE PUBLICATIS (1) Floquet,.; M Kadmi, C.; Perahia, D.; Gagne, D.; Bergé, G.; Marie, J.; Banères, J.-L.; Galleyrand, J.-C.; Fehrentz, J.-A.; Martinez, J. J. Mol. Biol. 2010, 395, 769. (2) Louet, M.; Perahia, D.; Martinez, J.; Floquet,. J. Mol. Biol. 2011, 411, 298. (3) Louet, M.; Charlier, L.; Martinez, J.; Floquet,. J. Chem. Inf. Model. 2012, 52, (4) Louet, M.; Martinez, J.; Floquet,. PLoS Comput. Biol. 2012, 8, e (5) Mary, S.; Damian, M.; Louet, M.; Floquet,.; Fehrentz, J.-A.; Marie, J.; Martinez, J.; Banères, J.-L. Proc. atl. Acad. Sci. U. S. A. 2012, 109, (6) Louet, M.; Karakas, E.; Perret, A.; Perahia, D.; Martinez, J.; Floquet,. FEBS Lett. 2013, 587, (7) Charlier, L.; Louet, M.; Chaloin, L.; Fuchs, P.; Martinez, J.; Muriaux, D.; Favard, C.; Floquet,. Biophys. J. 2014, 106, 577.

38

39 Abstracts For Poster Presentations

40 Synthesis of novel 3 -methyl-5 -norcarbocyclic nucleoside phosphonates as potential antiviral agents Jean-Pierre Uttaro, Christophe Mathé and Christian Périgaud Institut des Biomolécules Max Mousseron (IBMM), Université Montpellier 2, cc 1705, Place E. Bataillon, Montpellier, 34095, France [email protected] - web page: effecteurs- ucleoside analogues are an important class of drugs in the treatment of viral diseases. 1 evertheless, drug resistance and side effects have prompted to the development of new structurally modified nucleosides. Among them, phosphonate nucleoside analogues have demonstrated potent antiviral activities owing to the interactions with the nucleotide-converting enzymes as well as with their target viral polymerases. R a P a R' R, R' = H, H 2, H, H Figure 1. Structure of novel 3 -methyl-5 -norcarbocyclic nucleoside phosphonates. In this work, we report on the syntheses of novel 3 -methyl-5 -norcarbocyclic nucleoside phosphonates as potential antiviral agents (Figure 1). 2 1 De Clercq, E., Biochem. Pharmacol. 2013, 85, Uttaro, J.-P.; Broussous, S.; Mathé, C.; Périgaud, C. Tetrahedron 2013, 69,

41 2, 3 -Dideoxy-2 -fluoro-3 -(hydroxyimino), 3 -(methoxyimino) and 3 - (hydroxyamino) pyrimidine nucleoside derivatives Christophe Mathé, Ahmed Khalil and Christian Périgaud Institut des Biomolécules Max Mousseron (IBMM), Université Montpellier 2, cc 1705, Place E. Bataillon, Montpellier, 34095, France [email protected] - web page: effecteurs- ucleoside analogues play an important role in the treatment of viral diseases 3 and cancers. 4 In the search for new bioactive analogues, structural modifications of the heterocyclic base and/or on the sugar moiety of natural nucleosides can be envisioned. In this regard, introduction on the glycon moiety of an electron-withdrawing fluorine atom has been applied in the design of such analogues and led to compounds with significant biological activities. 5 Moreover, modifications of 2 - or 3 - position of nucleosides with others functionalities, such as hydroxyimino or hydroxyamino groups have also produced a variety of compounds with interesting biological activities. 6 Surprisingly, no work has been reported regarding the synthesis of compounds combining both modifications on the sugar moiety, i.e. presence of a fluorine atom and hydroxyimino, methoxyimino or hydroxyamino type functionalities. X X H H F R R = H, X = H R = Me, X = H R = Me, X = H 2 R = H, X = H 2 Figure H F H X = H X = H 2 2,3 -dideoxy-2 -fluoro-3 -(hydroxyimino), 3 -(methoxyimino) and 3 -(hydroxyamino)pyrimidine nucleosides In this work, we report on the synthesis of 2,3 -dideoxy-2 -fluoro-3 -(hydroxyimino), 3 - (methoxyimino) and 3 -(hydroxyamino)pyrimidine nucleosides (Figure) as well as the results of their biological evaluations. 7 3 De Clercq, E., J. Med. Chem. 2010, 53, Galmarini, C. M., Popowycz, F., Joseph, B., Curr. Med. Chem. 2008, 15, Liu, P., Sharon, A., Chu, C. K., J. Fluorine Chem. 2008, 129, Fedorov, I., Kazmina, E. M., Gurskaya, G.V., Jasko, M. V., Zavodnic, V. E., Balzarini, J., De Clercq, E., Faraj, A., Sommadossi, J. P., Imbach, J. L., Gosselin G., J. Med. Chem. 1997, 40, Khalil, A., Mathé, C., Périgaud, C., Eur. J. rg. Chem. 2012,

42 Detection of Intact Sulfated Peptides using MALDI-TF Mass Spectrometry Sonia Cantel, 1 Michael Smietana, 1 Luc Brunel, 1 Jean-Jacques Vasseur, 1 Christine Enjalbal, 1 Jean Martinez, 1 1 Institut des Biomolécules Max Mousseron, UMR 5247, Universités Montpellier 1 et 2, Montpellier, Cedex 5, France; [email protected] Protein identification and characterization has become one of the central activities in proteomics. The biological activity of many proteins is regulated by the extent and positions of post-translational modifications (PTMs). More than 200 different PTMs have been described but only a minor fraction of those has been studied in detail. Indeed, most of these PTMs are lacking appropriate analytical methods that allow the sensitive, residue-resolved detection and localization of the modifications. Sulfation of tyrosine residues are key post-translational modifications in the regulation of various cellular processes. As such, the detection and localization of tyrosine sulfation is an essential step toward the elucidation of the physiological and pathological roles of this process. Mass spectrometry has been developed as a key technology for protein modification analysis and many studies on sulfopeptides are MALDI-based. Despite substantial advances, intact sulfated peptides are still difficult to detect due to the extreme lability of the sulfo-moiety. We aim to develop a methodology involving a non-covalent derivatizing agent and ionization enhancer of the sulfo-containing peptides (small molecule/ peptide system) allowing direct and specific detection of mono- and polysulfated peptides in reflectron positive mode by MALDI-TF MS. S. Cantel, L. Brunel, K. hara, C. Enjalbal, J.J. Vasseur, M. Smietana, J. Martinez, Proteomics, 2012, 12,

43 Dynamic Materials for the Recognition and Transport of ligonucleotides Camille Bouillon, Delphine Paolantoni, Yannick Bessin, Pascal Dumy, Sébastien Ulrich Institut des Biomolécules Max Mousseron (IBMM), UMR CRS-UM1-UM2-ESCM 5247 Glycochemistry and Molecular Recognition team Ecole ationale Supérieure de Chimie de Montpellier (ESCM) 8 rue de l Ecole ormale Montpellier Cedex 5 France [email protected] The success of gene therapies such as antisense and silencing technologies depends mainly on the development of effective non-viral solutions for delivering therapeutic oligonucleotides into cells. To date numerous cationic polymers and dendrimers have been developed for this purpose. Despite their effective complexation with oligonucleotides these materials display significant limitations: a) their strong association prevents the release of oligonucleotides, and b) their macromolecular size results in poor elimination from the body, leading to toxic side-effects. A general solution to these problems may be the development of smart materials that degrade into smaller components inside cells, thereby resulting in a safer and more effective delivery of oligonucleotides. In this communication we will report the design and synthesis of hybrid dynamic materials that combine cationic monomers for promoting complex formation with oligonucleotides and ethylene oxide moieties for enhancing biocompatibility. Polymer formation is achieved by acylhydrazone bond formation and monitored by DSY MR. Furthermore, we demonstrate that these materials effectively complex oligonucleotides by fluorescence displacement assay and gel retardation assays. Since acylhydrazones are acidsensitive these materials may be of interest for the selective delivery of oligonucleotides into acidic compartments such as cancer cells.

44 Synthesis of new iminosugar derivatives by photoinduced thiol-ene coupling and evaluation of their glycosidase inhibitory properties Renaud Zelli, Pascal Dumy, Alberto Marra Institut des Biomolécules Max Mousseron (IBMM), UMR 5247, Glycochemistry and Molecular Recognition team, Ecole ationale Supérieure de Chimie de Montpellier, 8 Rue de l Ecole ormale, Montpellier Cedex 5, France, [email protected] Since the first isolation of the natural occurring nojirimycin by S. Inouye in the 60s, the interest in iminosugar chemistry never stopped growing because of their ability to inhibit glycosidases, enzymes involved in the carbohydrate metabolism. 1 owadays, two iminosugars are on the drugs market, Miglitol (1), to treat type II diabetes, and Zavesca (2), to treat lysosomale storage disorders (Gaucher s disease). H H H H H H H H H 1 2 We have prepared a library of new imino-disaccharides taking advantage of the photoinduced radical thiol-ene coupling (TEC), a metal-free reaction widely used in the field of biomolecules, 2,3 but never exploited for the synthesis of iminosugar derivatives. Starting from α- or β-d-glycosyl thiols as well as C-glycosylpropyl thiols, a series of imino-disaccharides was obtained in very good isolated yields. The inhibition properties of these compounds towards various glycosidases have been evaluated. H SH n + H H n = 0, 3 n = 1-3 n H hυ (365 nm) MeH H S n H n H H (1) Compain, P.; Martin,. R., Iminosugars: from synthesis to therapeutic applications, Wiley, (2) Dondoni, A.; Marra, A. Chem. Soc. Rev. 2012, 41, (3) Dondoni, A.; Marra, A., 2013, In Click chemistry in glycoscience: ew developments and strategies, Wictzak, Z. J.; Bielski, R., Wiley, pp 45-75

45 Chemical and biological challenges to study carbohydrate-lectin interactions - Applications in medicine and cosmetics Veronique Barragan-Montero, April Marquick, Sabrina Houaidji, Simona Sippelli, Lea Mba Mintsa, abil Kadri, Jean-Louis Montero Glycochemistry and Molecular Recognition Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CRS-UM1-UM2, Ecole ationale Supérieure de Chimie de Montpellier, 8 rue de l Ecole ormale, Montpellier Cedex, France [email protected] The research projects are dedicated to carbohydrates: methodology for the preparation of mannose, rhamnose derivatives as well as heterosides and supramolecular carbohydrate structures for medicinal and cosmetic applications. Some examples are given herein. 1) Towards a better understanding of the role of the Mannose-6-phosphate receptor in angiogenesis The syntheses of a series of more than twenty mannose-6-phosphate (M6P) analogues using methodologies developed by our team have been carried out. A particular strategy involved a cyclic sulfate of mannose as a common precursor. ther strategies have been developed: Mitsunobu, mecanochemistry, sonochemistry, etc... Some examples of the synthesised M6P analogues are presented herein. The replacement of the phosphate head-group by analogues, mostly bioisosteres, was intended to provide a better understanding of the chemical factors involved in the modulation of angiogenic activities. Currently, we are developing multivalent platformes such as nanoparticles (Au, Fe, Si, C) and cyclodextrins. Applications: Angiogenesis, an anti-cancer target: Some M6P analogues have proven to be angiogenic inhibitors (CAM). These carbohydrates are non-toxic, and have been proven to prolong life-expectency on mice. Angiogenesis in cardiology: Heart failure describes the incapacity of the heart to provide sufficient pump action to distribute the necessary amount of oxygene and metabolites that the body requires. Pro angiogenic saccharides are proposed. The abilities of these proangiogenic saccharides will be evaluated for ischemic heart failure in cardiology. 2) Adaptation of the immuno-precipitation technique for the discovery and isolation of new lectins A new rhamnose-binding lectin has been discovered and obtained from human skin by adapting the immuno-precipitation technique. Applications in dermatology and cosmetics are being considered. References: - S. Combemale, J.-. Assam Evoung, S. Houaidji, R. Bibi, V. Barragan-Montero Synthesis and Angiogenic Activity of Gold anoparticles Decorated with Mannose-6-phosphate Analogues, Molecules, 2014, 19, J.-L. Montero, V. Barragan-Montero, J.-P. Moles, P. de Santa Barbara, S. Combemale ouveaux dérivés de mannopyranoside ayant une activité anticancéreuse FR 2009, 09/ /11/2009. PCT 2010/ , 10/11/ V. Barragan-Montero, A. Awwad, S. Combemale, P. de Santa Barbara, B. Jover, J.-P. Molès, J.-L. Montero Synthesis of Mannose-6-Phosphate Analogues and their Utility as Angiogenesis Regulators, ChemMedChem 2011, 6, J.-L. Montero, V. Barragan-Montero, J.-P. Moles, S. Combemale Procédé de synthèse par chimie verte de dérivés anti-angiogéniques du mannose FR 2010, A. Bussière, V. Barragan-Montero, K. umzil, J.-Y. Winum, J.-L. Montero Expeditious stereoselective synthesis of L-iminosugars precursors via a Mitsunobu reaction Syn. Lett. 2009, 1, V. Barragan-Montero, A. Bussiere, C. Clavel, L. Toupet, J.-L. Montero A ne-step Synthesis of a Carbasugar via a Wittig-Horner/Mitsunobu/Michaël Triple-Tandem Mechanism, Lett. rg. Chem. 2006, 3,

46 Molecular Innovation and Design of ew Drug Systems Targeting the Tumour Associated Carbonic Anhydrase CA IX. Mohamed-Chiheb SAADA, Joanna MBUMA, Jean-Louis MTER and Jean-Yves WIUM* Team: Glycochemistry and Molecular Recognition Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CRS-UM1-UM2 Bâtiment de Recherche Max Mousseron, Ecole ationale Supérieure de Chimie de Montpellier, 8 rue de l Ecole ormale, Montpellier Cedex, France. [email protected] The zinc enzymes carbonic anhydrases (CAs, EC ) are very efficient catalysts for the reversible hydration of carbon dioxide to bicarbonate and hence play an important physiological role. In humans, 16 different isozymes have been described, some of them being involved in various pathological disorders. Several of these isozymes are considered as drug targets, and the design of selective inhibitors is a long-standing goal that has captured the attention of researchers for 40 years and has led to clinical applications against different pathologies such as glaucoma, epilepsy, and cancer. Carbonic anhydrase IX is expressed under hypoxic conditions and participate to the acidification of the tumoral microenvironment of solid tumor, participating to the metastasis process. CA IX is now a validated antitumor target, and its inhibition with antibodies, sulfonamide, and coumarin inhibitors has been undoubtedly proven to reverse the effect of tumor acidification, leading to the inhibition of the cancer cells growth in vivo. In this poster will be presented some of the results obtained in this research field by our group. Rami M, Dubois L, Parvathaneni K, Alterio V, van Kuijk SJ, Monti SM, Lambin P, De Simone G, Supuran CT, Winum JY. J Med Chem ov 14;56(21): Saada MC, mbouma J, Montero JL, Supuran CT, Winum JY. Chem Commun (Camb) Jun 25;49(50): McDonald PC, Winum JY, Supuran CT, Dedhar S. ncotarget Jan;3(1): Touisni, Maresca A, McDonald PC, Lou Y, Scozzafava A, Dedhar S, Winum JY, Supuran CT. J Med Chem Dec 22;54(24):

47 Temperature, ph and salt dependent visco-elastic behaviour of Gelatin hydrogels Y. Kadam *, 1, 2, A. El Ghzaoui 1, C. Pochat-Bohatier 2, J. Sanchez 2 1 Artificial Biopolymers - Institute of Biomolecules Max Mousseron (IBMM), UMR- CRS- 5247, University of Montpellier 1,34093 Montpellier, France. 2 Institut Européen des Membranes (IEM), UMR-CRS-5635, Université Montpellier II, 2 place Bataillon, Montpellier, France [email protected] Abstract: The increased use of synthetic polymers has led to a high ecological problem due to their total nonbiodegradability. Thus, there is a vital need to develop renewable and environmentally friendly biobased polymeric materials. Gelatin is a natural polymer widely used in pharmaceutical, cosmetic, photographic, and food industries. In this work, an experimental study was conducted on gelatin hydrogels and the effect of temperature, ph, zeta potential, and ionic strength on visco-elastic properties carried out, in order to determine the conformational characteristic, and phase transition (sol-gel transition; Ts-g) behavior. Keywords: Gelatin, hydrogel, visco-elastic, ph, ionic strength

48 Study of interactions between oppositely charged dendrigraft poly-l-lysine and Human Serum Albumin by frontal analysis continuous capillary electrophoresis icolas Sisavath 1, Laurent Leclercq 1, Thomas Le Saux 2, Hervé Cottet 1 1 Institut des Biomolécules Max Mousseron, Montpellier, 34095, France; 2 Ecole ormale Supérieure, Paris, 75231, France. [email protected] Dendrigraft poly-l-lysine (DGL) are biomacromolecules of great interest for many applications including antibacterial activity, drug delivery, gene therapy and production of antibodies. As human serum albumin (HSA) is the most abundant serum protein, the study of interactions between these two compounds is crucial for the use of DGL in drug or gene delivery systems. The present work aimed at studying the interaction between HSA and different generations of DGL under physiological conditions. To meet this end, a separative analytical method was developed for overcoming the adsorption phenomenon of one of the two oppositely charged species onto capillary wall. Frontal analysis continuous capillary electrophoresis was performed on a polycationic modified capillary and a co-pressure was adjusted for selectively introducing the free DGL from the DGL/HSA mixtures. The binding constants and the stoichiometry of the interaction were successfully determined using this low-consumption method [1]. Results obtained for the five generations of DGL (G1 to G5) showed an increase of the binding constants with the generation number. In contrast, a decrease of the DGL/HSA stoichiometry and of the cooperativity was observed with increasing generation number. G1 and G2 generations interacted with HSA with high cooperativity (Hill exponent ~ 2). G3 and G4 generations interacted with 2:1 DGL/HSA stoichiometries with only slight cooperativity for G3 (Hill exponent ~ 1.3) and with non cooperativity for G4 (2 independent sites). For G5, a 1:1 stoichiometry was found. These findings were in good agreement with the increase of ligand (DGL) size, the increase of ligand-ligand repulsion and the localization of two interaction sites on the HSA. [1]. Sisavath, L. Leclercq, T. Le Saux, F. ukacine, H. Cottet, J. Chromatogr. A 1289 (2013)

49 Determination of Polymer log D Distributions by Micellar and Microemulsion Electrokinetic Chromatography Xiaoyun Jin 1, Laurent Leclercq 1, Hervé Cottet 1 1 Institut des Biomolécules Max Mousseron (UMR 5247 CRS - Université de Montpellier 1 - Université de Montpellier 2), place Eugène Bataillon CC 1706, Montpellier Cedex 5, France @qq.com The characterization of the hydrophobicity of polymer compounds in solution remains a challenging issue of importance, especially for biomedical or pharmaceutical applications. In this work, for the first time, the log D distributions of statistical cationic polypeptides in the (-1 ; +3) range were characterized using micellar or microemulsion electrokinetic chromatography at physiological ph (7.4). The log D distributions of the polymer samples were obtained from the electrophoretic/chromatographic retardation of the polymer derivatives in presence of neutral micelles (or neutral microemulsion), using small cationic molecules of -blockers for calibration. Separating electrolytes were based on a TRIS-chloride buffer containing a neutral surfactant (polyoxyethyleneglycol dodecyl ether) for the formation of micelles (in water) or microemulsion (in water/n-pentanol mixture). The log D distributions obtained at ph 7.4 using this method were in good agreement with the chemical structures of cationic polypeptides: poly(lys, phe) 1:1>poly(lys, tyr) 1:1>poly(lys, trp) 4:1> poly(lys, ser) 3:1>poly(L-lysine), where x:y represents the molar ratio of each amino acid in the copolymer. Weight average octanol-water log D values and the dispersion of the log D distribution were also defined and determined for each polymer sample. Knowing the importance of the distributions in chemical composition and in molar mass on the properties of application of polymer samples, the opportunity to characterize the distribution in hydrophobicity seems very attractive. For example, in the pharmaceutical / biomedical field, it is known that the introduction of hydrophobic groups in polycations generally improves the transfection efficiency of gene delivery polyplexes into cells.

50 Potentiometric Investigation of Acido-Basic Behaviour and Complexation by Copper(II) Ions of Dendrigraft Poly-L-Lysine (DGL) in Aqueous Solution Jean-Christophe RSSI 1, Laurent BITEAU 1, Ling Phuong LUG 2, André DERATAI 2, Barbara MARET 1,3, Clément FAYE 3. 1 Institut des Biomolécules Max Mousseron UMR CRS 5247 CRS, University of Montpellier-1, University of Montpellier-2, CC 17006, Place E. Bataillon, F Montpellier cedex 5, France. 2 Institut Européen des Membranes IEM, UMR 5635 CRS, ESCM, Université Montpellier 2, Place E. Bataillon, F-34095, Montpellier, France. 3 Colcom, Cap Alpha, Avenue de l Europe, Clapiers, Montpellier cedex 09, France [email protected] Dendrigraft poly-l-lysines (DGL), discovered in our research group and currently commercialised by Colcom, are dendritic cationic polypeptides prepared in a straightforward and sustainable manner by iterative polymerization of ε-trifluoroacetyl-l-lysine--carboxyanhydride in aqueous medium.[1] DGL can be easily functionalized [2] and used for bio-applications such as drug or gene delivery,[3,4] transport through cellular membranes [5] or as nanosized magnetic resonance imaging contrast agents.[6] In view of future biological applications, a good knowledge of DGL physico-chemical properties in aqueous solution, such as protonation/dissociation extent, effective charge,[7] chelating affinity, etc, is necessary to understand their interactions with biomolecules [8] or with transition metal ions involved in metabolic processes. DGL protonation and Cu 2+ complexation, were investigated by GE and ISE potentiometry in presence of various Cu 2+ concentrations, with comparison to linear poly-l-lysines (α- / ε-pll), potentiometric / titration data being analyzed by different methods (Henderson-Hasselback, Bjerrum, Scatchard). Dissociation vs. ph profiles showed the presence of two distinct apparent pk A families, most probably related to α- and ε- free amino groups both present in DGLs; this feature also possibly explains the observed enhanced Cu 2+ complexation ability of DGL compared to α-pll. References [1] Collet, H.; Souaid, E.; Cottet, H.; Deratani, A.; Boiteau, L.; Dessalces, G.; Rossi, J.-C.; Commeyras, A.; Pascal, R. Chem. Eur. J. 2010, 16, [2] Rossi, J.-C.; Boiteau, L.; Collet, H.; Mbondo Tsamba, B.; Larcher,.; Pascal, R. Tetrahedron Lett. 2012, 53, [3] Liu, Y.; Li, J.; Shao, K.; Huang, R.; Ye, L.; Lou, J.; Jiang, C. Biomaterials 2010, 31, [4] Huang, R.; Liu, S.; Shao, K.; Han, L.; Ke, W.; Liu, Y.; Li, J.; Huang, S.; Jiang, C. anotechnology 2010, 21, [5] Tsogas, I.; Theodossiou, T.; Sideratou, Z.; Paleos, C.M.; Collet, H.; Rossi, J.-C.; Romestand, B.; Commeyras, A. Biomacromolecules 2007, 8, [6] gawa, M.; Regino, C.A.S.; Marcelino, B.; Williams, M.; Kosaka,.; Bryant, L.H.; Choyke, P.L.; Kobayashi, H. Bioconjugate Chem. 2010, 21, [7] Ibrahim, A.; Koval, D.; Kasiska, V.; Faye, C.; Cottet, H. Macromolecules 2013, 46, [8] Sisavath,.; Leclercq, L.; Le Saux, T.; ukacine, F.; Cottet, H. J. Chromatogr. A 2013, 1289,

51 ATCs, new thiazole-based γ-aminoacid building-blocks for the synthesis of helical foldamers [1] Loïc Mathieu, 1 Baptiste Legrand, 1 Cheng Deng, 2 Lubomir Vezenkov, 1 Emmanuel Wenger, 3 Claude Didierjean, 3 Muriel Amblard, 1 Marie-Christine Averlant-Petit, 2 icolas Masurier, 1 Vincent Lisowski, 1 Jean Martinez, 1 Ludovic T. Maillard 1 1 Institut des Biomolécules Max Mousseron, CRS-UM1-UM2 UMR 5247, Faculté de Pharmacie, 15 av. Charles Flahault, Montpellier, France 2 Laboratoire de Chimie Physique Macromoléculaire, CRS-IPL UMR 7568, Université de ancy, ancy, France 3 Laboratoire de Cristallographie, Résonance Magnétique et Modélisation, UMR 7063 CRS Université de Lorraine, Vandoeuvre-lès-ancy, France [email protected] A promising rational design approach for the discovery of protein-protein interaction inhibitors is centered on the design of peptidic or non-peptidic oligomers with conformational rigidity and proteolytic stability that reproduce key elements of protein secondary structures. Among the different strategies to mimic secondary structure elements like turns, helices and sheets, the foldamer approach has rapidly increased. Foldamers are defined as "oligomers that fold into a conformationally ordered state in solution, whose structures are stabilized by a collection of non covalent interactions between adjacent monomer units" [2]. -peptides are probably the most extensively studied foldamers. The success of these compounds is a result of their resemblance to -peptides as well as the diverse range of stabilized secondary structures that they could adopt. -peptides have been studied to a minor extent and only a few examples have been reported so far [3]. However, while -peptides contain more degrees of freedom than -peptides because of the additional carbon atom per backbone subunit, they also proved to fold into predictable conformations. Herein, we report the design of a new family of highly constrained thiazole-based -amino acids. We demonstrate by MR and X-ray crystallography studies that their corresponding oligomers could adopt a definite three-dimensional 9-helix structure both in solution and at the solid state stabilized by periodic intramolecular hydrogen bonds. These features resulted from the planar conformation of the C- C- C-C() torsion angle imposed by the thiazole heterocycle. [1] FmocH R 1 CH R 1 φ θ ψ FmocH ζ S H R 2 R 4 H R 1 S H R 1 S H R 1 S H R 1 S R 3 R 2 R 2 R 2 n R 2 Cristal structure and MR studies [1] Mathieu et al. Angew. Chem. Int. Ed., 2013, 52, 6006 [2] Appella et al, JACS, 1996, 118, ; Hill et al. Chem Rev, 2001, 101, [3] Bouillere et al. Amino Acids, 2011, 41, 687

52 ew Tools for Blood-Brain Barrier Drug Delivery: Design of ew Peptide-Based Vectors Jean-Daniel Malcor, 1, * adine Payrot, 2 Jean Martinez, 1 Michel Khrestchatisky, 2 Patrick Vlieghe, 2 Vincent Lisowski, 1 a- Institut des Biomolécules Max-Mousseron (IBMM, UMR5247 CRS), Universités Montpellier I et II, UFR des Sciences Pharmaceutiques et Biologiques, 15 avenue Charles Flahault, Montpellier Cedex 5, France. VECT-HRUS S.A.S., Faculté de Médecine Secteur ord, CS80011, Boulevard Pierre Dramard, Marseille Cedex 15, France. Drug delivery to the brain is hindered by the presence of the blood brain barrier (BBB). To accomplish the task of nutrient transport, the brain endothelium is endowed with various transport systems, including receptor-mediated transcytosis (RMT). This system can be used to shuttle therapeutics into the brain as a non-invasive manner. In this field, members of the low density lipoprotein receptor (LDLR) family are relevant as drug delivery systems. The main goal of this project is dedicated to the development of new peptide-based ligands of LDLR as potential BBB-vectors. The initial screening of a phage-display library directed to LDLR led to the identification of hits such as a cyclic 15-mer peptide with high in vitro affinity. A chemical optimisation led to the discovery of a new lead peptide, which brain uptake and in vivo pharmacological effects was measured. These first results were then assessed by biphotonic microscopy imagery.

53 Integrated Electrophoretic Microsystem for the Quality Control of conjugated PolyPeptides Gaëlle Coussot 1, Yoann Ladner 2, Catherine Perrin 3 1,2,3 IBMM, UMR 5247, University of Montpellier, 34093, France [email protected] Polypeptides (PP) are emerging as new drug substances or drug carriers. The PP structures can contain small active substances such as neurotransmitters, aminoacids or vitamins covalently linked to their backbone. PP can be synthetic non immunogenic homopolymers or highly specific proteins such as monoclonal antibodies. These novel PP therapeutic compounds have promising prospects in treating neurodegenerative diseases (Rheumatoid Arthrisis, Multiple Sclerosis) or others autoimmune pathologies. The characterization of PP compounds (consisting for instance in the determination of the mass percent ratio of small active substances) is required to allow marketing authorization of the PP compounds. However, characterization remains challenging due to the high polydispersity of the PP media. ne way to simplify PP-compounds is to undergo an enzymatic digestion step prior to the analysis by appropriate separation methods (HPLC, CE). In this context, the potential of an integrated on-line miniaturized digestion step (in-capillary digestion) before Capillary Zone Electrophoresis (CZE) separation has been investigated for the study of various PP compounds including therapeutic antibodies. Principles and first results of these both rapid and low operating costs microanalyses will be presented and discussed.

54 Fragment-Based Identification of a Locus in the AR-Sec7 Domain for the Design of Protein-Protein Interaction Inhibitors. François Hoh 3, Jad Rouhana 1, Corinne Henriquet 2, Martine Pugnière 2, André Padilla 3, Alain Chavanieu 1 (1) Institut des Biomolécules Max Mousseron UMR 5247 CRS, UM1, Montpellier, France. (2) Institut de Recherche en Cancérologie de Montpellier CRLC, ISERM U896, UM1, Montpellier, France (3) Centre de Biochimie Structurale UMR5048, ISERM 1054, UM1, Montpellier, France ABSTRACT: Low molecular weight G proteins of the Arfs family are implicated in a number of crucial cellular processes. Their GDP to GTP nucleotide exchange is controlled by ucleotide Exchange Factors (GEF), such as Arno, bearing a conserved Sec7 domain that catalyzes GDP dissociation from, and GTP binding to, their substrate Arfs. Among the six mammalian Arfs that control membrane traffic and organelles structures, Arf1 activation was recently reported to play a role in cancer progression and gastric carcinoma, and is a potential target for drug development. In our study, we developed a Fragment-Based Drug Design (FBDD) approach to establish a fragment binding mode to rationally design an inhibitor of the protein-protein interaction between Arf1 and the catalytic Arno-Sec7 domain. Initial selection of fragments was performed through a virtual screening approach of a library of ~3000 fragments, within small pockets on the Sec7 surface at level of interaction hotspots. The 33 selected fragments were evaluated for their potency to interfere with the nucleotide exchange catalyzed by Arno without bearing a promiscuous aggregate behavior. Using SPR and MR, the direct binding of three compounds to Arno, but not Arf1, was demonstrated. Then the mode of binding of one fragment was solved in complex with Arno-Sec7 domain by X-ray crystallography, and more active related compounds were then found. At last, the contribution in the fragment identification of the virtual screening tool, as well as the influence of the protein surface configuration, were investigated, which underlined the role of stable and transient pockets in the discovery and the binding of compounds. These results provide the first evidence at a structural level that Sec7 domains can be targeted by small organic compounds that shall guide competitive inhibitors rational drug design of the Arno activated Arf1 enzymatic activity.

55 A versatile total synthesis of E,E,Z-docosatrienes Balas L, Dayaker G, Durand T Institut des Biomolécules Max Mousseron, (IBMM, UMR5247 CRS-UM1-UM2-ESCM), Faculté de Pharmacie, 15 av. C. Flahault, BP 14491, Montpellier Cedex 5 (France) [email protected] The neuroprotectin D1 (PD1) is a bioactive endogenous dihydroxylated and non-cyclic metabolite of docosahexaenoic acid (DHA, C22:6 n-3). 1 H 17S Z E E H 10R H PD1 PD1 was shown to participate to the active process of resolution of inflammation. It reduces neutrophil accumulation and infiltration, and it is implicated in the return to cellular homeostasis, included wound healing. Since inflammation is a very complex multi-factorial process which plays a crucial role in many major current diseases such as Alzheimer s disease, atherosclerosis, cancers, cardiovascular diseases, chronic asthma, arthritis, etc., further investigation of this polyunsaturated metabolite is of great interest. However, for lack of standards in sufficient quantities, detection and quantification of this new endogenous lipid mediator in enflamed tissues, but also studies on the mechanism of its action (receptors, location, etc.) cannot be performed efficiently. Effectively, it is not commercially available and extractions of biological tissues provide tiny amounts only, thus restricting the number and the types of biological experiments. Furthermore, it is noteworthy that the ex-vivo enzymatic synthesis (ex vivo) starting from docosahexaenoic acid (DHA) and lipoxygenases did not yield to the expected molecule PD1 but to an isomer (called PDX) showing a E,Z,E-triene unit instead of the desired Z,E,E-triene unit. 2,3 Thus, we performed a total synthesis of a PD1 epimer using a flexible strategy that can give access to different diastereoisomers, isomers or analogs. The proposed strategy features Wittig olefinations, alkyne preparation, hydrometalations, Sonogashira cross coupling reactions. References (1) Serhan, C..; Petasis,. A., Chem Rev 2011, 111, (2) Chen, P. et al., FEBS Lett 2009, 583, (3) Butovich, I. A., J Lipid Res 2006, 47, 854.

56 Total synthesis of alkenyle and enediol dihomo-isofurans as biomarkers of the oxidative stress Aurélien de la Torre 1, Camille ger 1, Thierry Durand 1, Jetty Chung-Yung Lee 2, Jean-Marie Galano 1 1 Institut des Biomolécules Max Mousseron, UMR 5247 CRS - Universités Montpellier 1 & Montpellier 2 - ESCM, Montpellier 2 The University of Hong Kong, School of Biological Sciences, Hong Kong xidative stress (S) is a biological phenomenon involved in many pathologies, specially neurodegenerative diseases. This phenomenon has an impact on polyunsaturated fatty acids (PUFAs) through a non enzymatic radical pathway to form oxygenated metabolites. Those metabolites can be quantified in the biological fluids to evaluate the S. Thus, adrenic acid (AdA, C22:4 n-6), the most abundant PUFA in the brain s white matter, is metabolized into compounds known as dihomo- Isofurans, recently discovered by our team and highlighted in pig brains. 1 Therefore those compounds could be great biomarkers of myelin-related diseases, such as the Rett syndrome. The whole of these compounds being a huge family (256 isomers), it seems interesting to develop a flexible strategy which would allow us to access a good part of those isomers. These compounds have a common tetrahydrofuranic core bearing one hydroxyl function and two lateral chains of diverse structures, which can be classified in two categories: alkenyle and enediol (Scheme 1). ur strategy is based on the late introduction of the lateral chains from two cyclic intermediates obtained through the cyclization of a 1,2,5-triol and a 1,2,4-triol to access the alkenyle and enediol structures respectively. These triols are obtained by regioselective monoprotection of a common diol intermediate synthesized in 7 steps starting from commercial trans- -muconic acid. H H H H Alkenyle type Enediol type H H CR CR P' Ac Et Ac P' Et P P H H H H P' P H P H Et P' Et H P H Et H H trans-β-muconic acid Scheme 1: Divergent strategy for the total synthesis of Isofuran-like compounds 1 A. de la Torre, Y. Y. Lee, C. ger, P. T. Sangild, T. Durand, J. C-Y. Lee, J-M. Galano, 2014, Angewandt Chem. Int. Ed. in press

57 ovel Anti-Arrhythmics of Human rigin derived from on-enzymatic Peroxidation of DHA J. Roy, 1 C. ger, 2. Touzet-Mercier, 1 J. Roussel, 1 J. Thireau, 1 A. Guy, 2 V. Bultel-Poncé, 2 J-M Galano, 2 T. Durand, 2 J-Y Le Guennec. 1 1 Inserm U1046 Universités Montpellier-1 et Montpellier-2. 2 Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CRS, ESCM, Universités Montpellier-1 et Montpellier-2. Since forty years, it is known that polyunsaturated fatty acids (PUFAs) of the n-3 series have cardioprotective effects by preventing cardiac arrhythmias. 1,2 The main PUFAs involved are eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The effects of n-3 PUFAs on cardiac function are still debated, notably because of the lack of information on the mechanisms involved. ur hypothesis is that, during an infarct, the generation of reactive oxygen species (RS) coming from oxidative stress (S) might be responsible for an oxidation of membrane-bound PUFAs. 3 The oxygenated metabolites thus generated might modulate the activity of ionic channels to exert antiarrhythmic effects. 4 We thus decided to investigate the influence of the non-enzymatic peroxidation of DHA and the anti-arrhythmic properties of oxygenated metabolites, to validate if the supposed antiarrhythmic properties of n-3 PUFAs are not due to their non-enzymatic oxygenated metabolites. 5-7 Thanks to our efficient and flexible strategy, several IsoPs and europs were successfully synthesized and studied. The proof of concept was validated both in vitro and in vivo, showing the anti-arrhythmic properties of some of the non-enzymatic oxygenated metabolites of DHA called europs. 8 References: [1] Bang H. et al. (1971) Lancet, 1, [2] Saravanan P. et al. (2010) Lancet, 376, [3] Jahn, U. et al. (2008) Angew. Chem. Int. Ed. 47, [4] Judé, S. et al. (2003) Br. J. Pharmacol. 139, [5] Le Guennec, JY. et al. European Patent 5 December EP PCT 4 december PCT/EP2013/ [6] ger, C. et al. (2010) Chem. Eur. J. 16, [7] Galano JM. et al. (2013) Prostaglandins & ther Lipid Med. 107, [8] Roy J. et al. (2014) ature, submitted

58 Design, synthesis and in vivo studies of new stable neurotensin analogues as potent antinociceptive agents. Roberto Fanelli (1), Adeline René (1), Pascal Tétreault (2), Mylène Lafrance (2), icolas Floquet (1), Philippe Sarret (2), Jean Martinez (1), Florine Cavelier (1) *. (1) IBMM, UM1-UM2-CRS UMR 5247, Montpellier, France (2) Department of physiology and biophysics, Faculty of medicine and health sciences, Sherbrooke, CAADA eurotensin (T) is a tridecapeptide which was first isolated from bovine hypothalamus. (a) This molecule exerts a variety of physiological effects as hypothermia, analgesia or antipsychotic properties. Structure-activity relationship studies showed that the C-terminal fragment of neurotensin, called T(8-13), was the minimal active sequence. As many peptides, neurotensin posses a short half-life time due to enzyme degradation. Indeed, electrophoresis analysis and plasmatic stability studies highlighted that specific enzymes, namely endoproteases, can affect three out of the five peptide bonds. In order to overcome neurotensin instability, we developed several T analogues using different approaches including unnatural amino acid incorporations, (b) peptide bond modifications and cyclisation. (c) ne of our objectives was to target the T-induced analgesic effect. Among the three receptor subtypes, Sarret et al (d) showed that selectivity toward TS2 receptor is a parameter that has to be considered in the analogue design strategy. Molecular modeling calculations have been launched to assist the improvement towards TS2 selectivity and helping the design of new analogues. A strategical synthetic approach was initiated to study structure-activity relationship of the active T(8-13) fragment leading to bioactive and resistant T-analogues. Synthesis, binding affinity as well as in vivo analgesic activity on acute and neuropathic pain in rats will be presented. Bibliographic references: (a) Carraway, R. E., Leeman S. E., (1973), J. Biol. Chem. 248: (b) Vivet, B., Cavelier, F., Martinez, J. (2000), Eur. J. rg. Chem. 5, (c) Bredeloux, P., Cavelier F., Dubuc, I., Vivet, B., Costentin, J., Martinez, J., (2008), J. Med. Chem., 6, (d) Roussy, G., Dansereau, M-A., Baudisson, S., Ezzoubaa, F., Belleville, K., Beaudet,., Martinez, J., Richelson, E., Sarret, P., (2009), Mol Pain., 5, 38.

59 Homopolysilaproline a new peptide with PPII structure Florine Cavelier 1 Charlotte Martin 1 Batiste Legrand 1 Aurélien Lebrun 2 Jean Martinez 1 1 IBMM, UM1-UM2-CRS UMR 5247, Montpellier, 34095, FRACE 2 LMP, Montpellier, FRACE [email protected] The importance of the left-handed polyproline II (PPII) helical conformation has recently become apparent. This conformation generally is involved in two important functions: protein-protein interactions and structural integrity. The PPII helix is believed to be the dominant conformation for many proline-rich regions of peptides and proteins. In particular PPII are major features of collagens. In our laboratory, an original proline analogue has been synthesized: the 4,4-dimethylsilaproline, denoted silaproline (Sip) 1. The presence of dimethylsilyl group confers to silaproline a higher lipophilicity as well as an improved resistance to biodegradation. Recently we developed a gram scale synthesis of enantiomerically pure Sip, requiring resolution of the racemate by chiral high performance liquid chromatography (HPLC)2. With this new starting material, our aim was to construct polysilaproline polymers to investigate physico chemical properties and modifications of PPII structure. In this study monodisperses homopolypeptides have been synthesized by peptide coupling in solution, polydisperses homopolypeptides have been prepared by ring opening polymerization of - carboxyanhydrides (RP). These peptides have been characterized by RM, MALDI-Tof and circular dichroism. [1] Vivet B., Cavelier F., Martinez J. (2000), Eur. J. rg. Chem., 5 : [2] Martin C., Vanthuyne., Miramon H., Martinez J., Cavelier F. (2012), Amino Acids, 43:

60 Biomolecules of Tea Fatiha Sebih a, b, Janique Guiramand a, Catherine Cohen-Solal a, Michel Vignes #a and Valerie Rolland #a* «xidative stress and neuroprotection» Team a IBMM-UMR5247, CRS, UM2, UM1, Place E. Bataillon, Montpellier Cédex5, France. b Laboratoire de Synthèse rganique Appliquée LSA, Université d ran, Département de Chimie, Bp 1524 El M aouer, ran 31000, Algérie [email protected] & [email protected] It is now accepted that green tea consumption is beneficial to human health. Most of these health benefits are associated with green tea antioxydant activity. Indeed, potent antioxydants belonging to the catechin family, including epi-gallocatechin-gallate (EGCG) are encountered in green tea. evertheless, other molecules, such as theanine, a unique amino-acid found in green tea, may also contribute to its beneficial effects. With regards to the central nervous system (CS), both EGCG and theanine have proved to be neuroprotective in various models of brain injury, such as ischemia and neurodegenerative diseases. This is due to their ability to cross the blood-brain barrier. In addition, both molecules seem to exert rapid and long-lasting protective effects. While their long-lasting effects involve genomic actions, rapid effects appear to be restricted to action on neuronal membranes such as the modulation of neurotransmitter receptors. We illustrate here rapid CS actions of EGCG and theanine with the demonstration of the anxiolytic activity of EGCG, via the modulation of GABA A receptors and the co-agonist activity of L- theanine towards glutamate -methyl-d-aspartate (MDA) receptors. In this line, derivatives synthesized from the theanine backbone are a new family of MDA receptor ligands. References: «Anxiolytic properties of green tea polyphenol ( )-epigallocatechin gallate (EGCG)» Michel Vignes, Tangui Maurice, Fabien Lanté, Magali edjar, Karen Thethi, Janique Guiramand, Max Récasens. Brain Research Volume 1110, Issue 1, 19 September 2006, Pages «Tea Health and Disease Prevention» Ed V.R.Preedy ELSEVIER- Michel Vignes 2013, p1399. ISB Synthesis and functional characterization of novel MDA receptor ligands based on L-Theanine backbone Fatiha Sebih, Mohammed Abouazza, Salima Bellahouel, Aicha Derdour, Marie Céleste de Jésus Ferreira, Janique Guiramand, Catherine Cohen-Solal, Gérard Barbanel, Jean Martinez, Michel Vignes and Valerie Rolland. Bioorg.Med.Chem., 2014 under review.

61 Laser desorption ionization mass spectrometry of peptides on amorphous CHCA-functionalized silica Clément Fleith, 1 Jérémie Ciccione, 1 Sonia Cantel, 1 Gilles Subra, 1 Jean Martinez, 1 Ahmad Mehdi, 2 Christine Enjalbal 1 1 Institut des biomolécules Max Mousseron, UMR 5247, Universités Montpellier 1 et 2, Montpellier, Cedex 5, France; 2 Institut Charles Gerhardt, UMR 5253, Université Montpellier 2, Montpellier Cedex 5, France [email protected] In our previous investigations (1-3), we reported the use of silica materials for tryptic digest analysis in as laser desorption/ionization mass spectrometry (LDI-MS). In order to increase the UV laser absorption capacity of the inert substrates, we designed new materials combining properties from organic and silica matrices. We decided to functionalize and hyper-functionalize silica with the popular MALDI matrix α-cyano-3-hydroxy-cinnamic acid (CHCA) through different classic sol-gel chemistries. The driving force of our work was to increase the organic/inorganic ratio of our silica based materials, in order to improve the UV photon absorption prerequisite for efficient LDI mechanism. We succeeded in preparing an amorphous silica material with a high content of CHCA (up to 1.3 mmol.g -1, around 40 % in weight from TGA and elementary analyses measurements) through a 5 steps preparation protocol. We report applications of such new hybrid organic/inorganic silica based materials as laser desorption/ionization promoting substrates for high-throughput identification of peptides (4). To assess the performances of the LDI method, in terms of sensitivity and ionization discrimination, the detection of small synthetic peptides ( Da) conditioned as mixtures was investigated. The peptide behaviors were compared with the references, CHCA for organic matrix, and amorphous silica for inert matrix. As expected, this hybrid material provided also hybrid results between organic and inert matrices. This new matrix was also evaluated to perform efficient MS/MS sequencing at low peptide concentrations. 1. M. Dupré, S. Cantel, J.-. Durand, J. Martinez and C. Enjalbal, Analytica Chimica Acta, 2012, 741, M. Dupré, Y. Coffinier, R. Boukherroub, S. Cantel, J. Martinez and C. Enjalbal, Journal of Proteomics, 2012, 75, M. Dupré, C. Enjalbal, S. Cantel, J. Martinez,. Megouda, T. Hadjersi, R. Boukherroub and Y. Coffinier, Analytical Chemistry, 2012, 84, C. Fleith, S. Cantel, G. Subra, A. Mehdi, J. Ciccione, J. Martinez, C. Enjalbal, Analyst, 2014, in press.

62 Robust Helix Formation in a ew Family of Constraint β-bicyclic Amino Acid ligoureas Christophe André, 1 Baptiste Legrand, 1 Emmanuel Wenger, 3 Claude Didierjean, 3 Marie Christine Averlant-Petit, 2 Jean Martinez, 1 Monique Calmès, 1 Muriel Amblard 1 1 Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CRS-Université Montpellier 1 et 2, 15 Avenue Charles Flahault, Montpellier, France. 2 Laboratoire de Chimie-Physique Macromoléculaire, ancy Université, CRS, 1 rue Grandville, ancy Cedex 1, France. 3 Laboratoire de Cristallographie, Résonance Magnétique et Modélisation, ancy Université, UMR7036 CRS-UHP, Boulevard des Aiguillettes, Vandoeuvre-Lès-ancy Cedex, France. [email protected] Among the foldamers based on natural peptide sequences, γ-peptides have not received so much attention. An elegant way for the construction of γ-peptide mimetics was the use of an urea linkage. In this study, our aim was to design new oligomers able to display a well-defined secondary structure combining the benefit of a conformationally constrained bicyclic β-amino acid and the bifidic hydrogen bonds stabilization brought by urea links. We selected the (S)-aminobicyclo[2.2.2]octane-2-carboxylic acid [(S)-ABC] as constrained motif. To study the progressive folding of the corresponding homooligoureas, we prepared BAC oligoureas of different lengths and investigated their conformational preference by a combination of MR and CD spectroscopy and X-ray crystallography.

63 Mixed ligoureas Based on Constrained Bicyclic and Acyclic β-amino Acids Derivatives: n the Significance of the Subunit Configuration for Folding Christophe André, 1 Baptiste Legrand, 1 Laure Moulat, 1 Emmanuel Wenger, 3 Claude Didierjean, 3 Marie Emmanuel Aubert, 3 Christine Averlant-Petit, 2 Jean Martinez, 1 Monique Calmès, 1 Muriel Amblard 1 1 Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CRS-Université Montpellier 1 et 2, 15 Avenue Charles Flahault, Montpellier, France. 2 Laboratoire de Chimie-Physique Macromoléculaire, ancy Université, CRS, 1 rue Grandville, ancy Cedex 1, France. 3 Laboratoire de Cristallographie, Résonance Magnétique et Modélisation, ancy Université, UMR7036 CRS-UHP, Boulevard des Aiguillettes, Vandoeuvre-Lès-ancy Cedex, France. [email protected] The ability of β- and γ-amino acid oligomers to adopt protein-like secondary structures has emerged as a particular attractive field of research to design natural structural mimics of biomolecules. In this study, we investigated the propensity of the constrained bicyclic amino carbamoyl BAC residue to induce helical structures when combined with acyclic (S)-β 3 -amino acid derivatives. Circular dichroism (CD), MR and X-ray crystallography studies of the BAC-based mixed systems showed that the alternating heterochiral R/S sequences form a stable left-handed 2.5-helix in contrast to the mixed S/S-oligoureas.

64 A new approach for the synthesis of peptide-based polymer Soultan, A 1 ; Jebors, S 1 ; Cicionne J 1 ; Enomoto, H; ottelet, B; Lebrun, A; Mehdi, A; Enjalbal, C; Dupre, M; Tailhades, J; Coudane, J; Martinez, J 1 ; Subra, G 1 ; Amblard, M 1 1 Faculté de pharmacie (Bât E, 3 ème étage) : 15, Avenue Charles Flahault, Montpellier, Cedex 5 [email protected] Peptides and polypeptides are attracting considerable interest in the field of biomaterials and biological sciences because of their biocompatibility and biodegradability properties. A common approach relies on post grafting of peptide sequences, using chemoselective reactions, on modified polymers. However, direct polymerization of large, synthetic peptides or macromolecules remains a challenge. In this context, we developed new strategies to polymerize well defined peptides. High molecular weight polymers can be prepared in both good yield and large quantity. The first one was the polymerization of α-amino -carboxy-α-amino acid anhydrides (CAs) which generated peptidebased branched polymers. For that, we developed a convenient and straightforward methodology to generate the CA activated species directly on the solid support. The activated peptide block was then released from the solid support to be engaged in the CA/Ring pening Polymerization (RP) reaction (Fig. 1) and gave comb polymer. The second strategy was to form Si--Si bond (siloxane) by poly-condensation of Si-H (silanol). This method need to introduce two silanol functions at the C and terminus of the peptide sequence or alternatively, on a -terminal Lysine. Polymerization was then carried out in phosphate buffer to give linear or comb-like polypeptides (Fig 2). Figure 1 : Linear polymer Figure 2 : Comb polymer 1. Kricheldorf H, et al (1990) Polypeptides pp Kricheldorf HR (1987), Springer, ew York 3. Hiroshi Enamoto (2013), Chem. Commun. 49, Verdie, P., et al., (2011), Chem-Asian J 6: Subra, G., et al., (2011), J Mater Chem 21:

65 Pipecolic linker for Peptide and pseudo-peptide synthesis icolas Masurier, a Pawel Zajdel, b Pierre Sanchez, a Pascal Verdié, a Maciej Pawlowski, b Muriel Amblard, a Jean Martinez, a Gilles Subra a a Institut des Biomolécules Max Mousseron, CRS UMR 524, Universités Montpellier I & II, Faculté de Pharmacie, 15 avenue Charles Flahault, Montpellier, France b Department of Medicinal Chemistry, Jagiellonian University Medical College, 9 Medyczna Street, Kraków (Poland) Peptides are widely considered as privileged structure to access to lead compounds in drug development. And nowadays, peptide based therapeutics exist for a wide variety of diseases. Despite their high potentials, peptides present major disadvantages: short half-life, rapid metabolism, poor oral bioavailability, etc. evertheless, development of pseudo-peptides by different types of chemical modifications can permit to avoid these disadvantages. Various peptidomimetic approaches used for the design and synthesis of peptide analogs with improved pharmacological and pharmacokinetic properties were proposed. In particular, solid phase synthesis of pseudo-peptides libraries is particularly powerful. Recently, we proposed a novel TFA-labile linker based on the pipecolic acid scaffold 1, which could be readily activated to anchor a range of nucleophiles. Its use was demonstrated for alcohol and amine side chain and -terminus anchoring of peptides. 1 We now report the use of this novel linker for peptides, pseudo-peptides synthesis and preparation of modified amino-acids on solid support. In particular, incorporation of a urea or a double bond could be easily achieved using this linker. Preparation of C-terminal hydrazide peptide, cyclic peptides and C-terminal pseudo-peptide alcohol was also demonstrated. H Et H 2 Insertion of double bond Me H 2 H H insertion of urea bond H 2 H 2 H 2 AM-PS resin 1 H * H H H H H H H H H H pipecolic linker H H H Cyclic peptide H H H H H 2 H H H H H Fmoc gem-diamino peptide H H H H C terminal hydrazino peptide H H H 2 H H H 2 C terminal peptide alcohol References 1. a) J. Martinez, P. Zajdel, M. Pawlowski, G. Subra. E Patent n ; b) P. Zajdel, G. omézine,. Masurier, M. Amblard M., M. Pawlowski, J. Martinez, G. Subra, Chem Eur J., 2010, 16(25), ; c) P. Zajdel,. Masurier, P. Sanchez, M. Pawlowski, A. Kreiter, G. omézine, C. Enjalbal, M. Amblard, J. Martinez, G. Subra, J. Comb. Chem., 2010, 12(5), ; d) d). Masurier, P. Zajdel, P. Verdié, M. Pawlowski, M Amblard., J. Martinez, G. Subra, Chem Eur J., 2012, 18,

66 Sustainable methods to make an amide bond Frédéric Lamaty, 1 Evelina Colacino, 1 Xavier Bantreil, 1 Thomas-Xavier Métro, 1 Pauline Petiot, 1 Clarence Charnay, 2 Lucy Puttergill, 1,2 Valérie Declerck, 1 Pierrick un, 1 Julien Bonnamour, 1 Thomas Reidon, 1 Jordi Sarpoulet, 1 Clément Fleith 1 and Jean Martinez 1 1 Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CRS-Universités Montpellier 1 & 2, Place Eugène Bataillon, Montpellier Cedex 05, France; 2 Institut Charles Gerhardt Montpellier (ICGM), UMR 5253 CRS-Université Montpellier II, Place E. Bataillon, Montpellier Cedex 05, France [email protected], The amide functional group is of high interest with respect to the structure of natural and non-natural products, bioactives molecules, drugs and peptides. Present in a molecule, the amide bond will provide unique polarity, solubility and interactions properties. General methods to form amides rely on the reaction of activated acid moieties with amines. Such methodologies carried out in classical solvents are generally expensive and generate a significant amount of waste. Various approaches based on technologies and reactions developed in our group will be presented. In order to expand the tool box of sustainable methods, some of the 12 principles of Green Chemistry were used as guidelines for our work. Herein will be described three different approaches : the synthesis of peptides in PEG-based ionic liquids, 8 the preparation of the amide bond in solvent-free condtions in a ball-mill 9 and the copper-catalyzed formation of amides starting from alcohols and amines Petiot, P.; Charnay, C.; Martinez, J.; Puttergill, L.; Galindo, F.; Lamaty, F.; Colacino, E. Chem. Commun. 2010, a) Declerck, V. ; un, P.; Martinez, J.; Lamaty, F. Angew. Chem. Int. Ed. 2009, 48, b) Métro, T.-X.; Bonnamour, J.; Reidon, T.; Sarpoulet, J.; Martinez, J.; Lamaty, F. Chem. Commun. 2012, 48, c) Bonnamour, J.; Métro, T.-X.; Martinez, J.; Lamaty, F. Green Chem. 2013, 15, Bantreil, X.; Fleith, C.; Martinez, J. and Lamaty, F. ChemCatChem 2012, 4, 1922.

67 Exploring frontiers of peptides synthesis through mechanochemistry Julien Bonnamour, Thomas-Xavier Métro, Jean Martinez and Frédéric Lamaty Institut des Biomolécules Max Mousseron (IBMM) UMR 5247 CRS-Universités Montpellier 1 et 2, Université Montpellier 2, Place Eugène Bataillon, Montpellier cedex 05, France. [email protected] Classical amides and peptides synthetic methods suffer from various limitations: - large amounts of reagents and solvents are needed - required solvents are often very toxic, - production of large quantities of waste, - low productivity, - side reactions due to highly diluted reaction media, - peptide size limitations due to aggregation We have recently shown that using mechanochemistry can help in solving these limitations. 1, 2 The use of high-energy ball-milling has allowed us to produce various peptides by treating stoichiometric amounts of activated amino acids (Boc-AA-Su and UCA) with amino acid alkyl ester salts in the presence of innocuous ahc 3 and EtAc. Di- to penta-peptides such as Leu-enkephalin were produced in high yields without requiring tedious chromatography purifications (Scheme 1). 2 Efficient synthesis of Boc-(Leu) 4 -Bn, which is highly prone to aggregation, let us envisage excellent abilities for this mechanically-assisted approach to enable the synthesis of other difficult and much longer peptide sequences. Mechanically-assisted peptide synthesis 1. Declerck, V.; un, P.; Martinez, J.; Lamaty, F., Solvent-Free Synthesis of Peptides. Angew. Chem. Int. Ed. 2009, 48, Bonnamour, J.; Métro, T.-X.; Martinez, J.; Lamaty, F., Environmentally benign peptide synthesis using liquid-assisted ball-milling: application to the synthesis of Leu-enkephalin. Green Chem. 2013, 15,

68 Solventless - And C-Protection of Amino Acids and Esters in a Ball-Mill Laure KERT, Frédéric LAMATY, Jean MARTIEZ, Evelina CLACI Institut des Biomolécules Max Mousseron, UMR 5247 CRS-UM1-UM2, Université Montpellier II, Place Eugène Bataillon Montpellier Cedex 5, France ; web page: [email protected] In our on-going work on mechanochemistry, 1 we developed an eco-friendly methodology for preparing - protected -amino acids and esters using ball-milling technology. This solvent-free methodology gave Boc-, Fmoc- and Z--protected amino acids. 1a A number of amino esters (alkyl, allyl ) was also obtained via C-activation of the amino acids. The method presents an improved environmental impact compared to the classical syntheses in solution. Different ball mills (vibration or planetary) were used, tuning the process parameters (grinding material, number of balls, frequency and rotation speed) according to the targeted protection. -protection R 2 = H Vibrational Ball-Mill PG H R 1 H Protecting Group (PG) = Fmoc, Z, Boc Planetary Ball-Mill R1 R H2 2 -protection Protecting group / Base R 2 = Me, t-bu PG H R1 R 2 C-protection/activation Chloroformates or (di)-carbonate derivatives / Base R 2 = Me, t-bu, Bn, Allyl, Su PG H R1 H 1) a) L. Konnert, A. Gauliard, F. Lamaty, J. Martinez, E. Colacino, ACS Sus. Chem. Eng. 2013, 1, ; b) E. Colacino, P. un, F. M. Colacino, J. Martinez, F. Lamaty, Tetrahedron 2008, 64, ; c) T. X. Métro, J. Bonnamour, T. Reidon, J. Sarpoulet, J. Martinez, F. Lamaty, Chem. Commun. 2012, 48, ; d) V. Declerck, P. un, M. J., F. Lamaty, Angew. Chem. Int. Ed. 2009, 48, ; e) J. Martinez, F. Lamaty, V. Declerck, W , 2008; f) A. Baron, J. Martinez, F. Lamaty, Tetrahedron Lett. 2010, 51, ; g) P. un, V. Pérez, M. Calmès, J. Martinez, F. Lamaty, Chem. Eur. J. 2012, 18,

69 Mechanochemical Synthesis of Hydantoins Laure KERT, Frédéric LAMATY, Jean MARTIEZ, Evelina CLACI Institut des Biomolécules Max Mousseron, UMR 5247 CRS-UM1-UM2, Université Montpellier II, Place Eugène Bataillon Montpellier Cedex 5, France ; web page: [email protected] Hydantoins are a class of well-known molecules with numerous biological activities. 1 The therapeutic properties of some hydantoin derivatives, such as phenytoin (antiepileptic), 2 ethotoin (anticonvulsant) 3 or nilutamide (antiandrogenic) 4 were demonstrated. In our on-going work on mechanochemistry, 5 we used ball-milling technology to prepare diverse hydantoins, including phenytoin and ethotoin, according to an easy and environment-friendly solvent-free procedure and a practical precipitation/filtration work-up. H H H H 2 CF 3 Phenytoin Ethotoin ilutamide Bibliographic references: (1) Meusel, M.; Gutschow, M. rg. Prep. Proced. Int. 2004, 36, (2) Dhanawat, M.; Banerjee, A. G.; Shrivastava, S. K. Med. Chem. Res. 2011, 21, (3) Kutt, H.; Harden, C. L. Handb. Exp. Pharmacol. 1999, 138, (4) Grosse, L.; Paquet, S.; Caron, P.; Fazli, L.; Rennie, P. S.; Belanger, A.; Barbier,. Cancer Res. 2013, 73, (5) a) L. Konnert, A. Gauliard, F. Lamaty, J. Martinez, E. Colacino, ACS Sus. Chem. Eng. 2013, 1, 9, ; b) E. Colacino, P. un, F. M. Colacino, J. Martinez, F. Lamaty, Tetrahedron 2008, 64, ; c) T. X. Métro, J. Bonnamour, T. Reidon, J. Sarpoulet, J. Martinez, F. Lamaty, Chem. Commun. 2012, 48, ; d) V. Declerck, P. un, M. J., F. Lamaty, Angew. Chem. Int. Ed. 2009, 48, ; e) J. Martinez, F. Lamaty, V. Declerck, W , 2008; f) A. Baron, J. Martinez, F. Lamaty, Tetrahedron Lett. 2010, 51, ; g) P. un, V. Pérez, M. Calmès, J. Martinez, F. Lamaty, Chem. Eur. J. 2012, 18,

70 HslVU of Leishmania and Trypanosoma, a mitochondrial proteasome-like complex as a potential therapeutic target Samanta K. 1, Kebe M.. 2, Mbang-Benet D.E. 3, Apicella V. 1, Payrot. 1, Lisowski V. 1, Kajava A. 2, Pagès M. 3, Martinez J. 1, Bastien P. 3, Coux. 2, Hernandez J.-F. 1 1 Institut des Biomolécules Max Mousseron, UMR 5247 CRS Universités Montpellier 1 et 2, Faculté de Pharmacie, Montpellier, France; 2 Centre de Recherches de Biochimie Macromoléculaire, UMR 5237 CRS, Universités Montpellier 1 et 2, Montpellier, France; 3 Département de Parasitologie-Mycologie, MeBFEA, UMR 5290 CRS 224 IRD, Université Montpellier 1, MIVEGEC, Montpellier, France. [email protected] It is urgent to develop less toxic and more efficient treatments for Leishmaniases and Trypanosomiases. We propose to target a proteasome-like complex, the HslVU protease, which is present in the parasite s single mitochondrion, essential for the growth of these organisms and has no analogue in the human host. riginally discovered in eubacteria, this complex is constituted by two central hexameric HslV protease rings sandwiched between two hexameric HslU ATP-ase rings. As HslV shares a similar enzymatic mechanism with the host proteasome, we propose to inhibit the assembly of the complex in order to be selective. According to studies on bacterial HslVU, 1,2 the C- terminal segment of HslU is essential in HslV activation and in complex assembly, therefore representing a privileged target. We produced recombinant HslV, which is inactive alone, and showed that a synthetic C-terminal HslU dodecapeptide was able to induce the digestion by HslV of a fluorogenic substrate that we developed. With this enzymatic test in hands, we started the characterization of the interaction of the C-terminal portion of HslU with HslV. We will present the results obtained with various series of analogues of the original C-terminal HslU peptide, including truncated forms, Ala scan, constrained analogues and multivalent constructions. The aim is to establish structural requirements, which could lead to high affinity and stable ligands able to inhibit the interaction between the HslU and HslV rings, obligatory for the degradation of proteins by the HslVU complex. We checked that HslV was inhibited by classical active-site directed proteasome inhibitors like bortezomib. Finally, we started to consider the mitochondrial addressing of C-ter HslU-derived peptides by mitochondria-penetrating peptides (MPP). Fluoroscein-labeled MPPs were synthesized and incubated with parasites. Fluorescence microscopy showed that some analogues entered the parasites but without specific accumulation in the mitochondrion.

71 HslU Hexameric ring ATPase C-ter HslU HslV Hexameric ring Protease HslV HslU 3D structure of HslVU from Haemophilus influenzae showing the interaction between the C-terminal segment of HslU and HslV. 3 C-terminal segments of HslU from bacteria and parasites are highly homologous. [1] Ramachandran R., Hartmann C., Song H. K., Huber R., Bochtler M. (2002) Proc. atl. Acad. Sci. USA, 99, [2] Seong I. S., Kang M. S., Choi M. K., Lee J. W., Koh. J., Wang J., Eom S. H., Chung C. H. (2002) J. Biol. Chem., 277, [3] Souza M. C., Trame C. B., Tsuruta H., Wilbanks S. M., Reddy V. S., McKay D. B. (2000) Cell, 103,

72 Fluorescent Biosensors of Cyclin-Dependent Kinases tools for probing relative abundance, activity and conformational dynamics in real time Camille Prével 1, Morgan Pellerano 1, Thi hu goc Van 2, Laetitia Kurzawa 3, Marion Peyressatre 1, Veronique Josserand 4, Jean-Luc Coll 4 & May C. Morris 1 * 1 Institut des Biomolécules Max Mousseron-UMR5247, Montpellier, France; 2 Bordeaux, France; 3 Grenoble, France ; 4 Institut Albert Bonniot ISERM U Grenoble, France * [email protected] Cell cycle progression is driven by a family of Cyclin-Dependent Kinases (CDKs), serine/threonine protein kinases whose sequential activation coordinates cell growth and division. CDK/cyclin levels and activities are frequently altered in human cancers, thereby contributing to sustain aberrant proliferation in cancer cells. Despite the oncological relevance and pharmacological attractivity of these heterodimeric kinases, there are no direct means of assessing their relative abundance and activities in living cells. Detection of CDK/cyclin kinases remains essentially limited to antigenic approaches following cell or tissue fixation, whilst activity measurements are limited to discontinuous, endpoint radioactive assays. We have developed a family of environmentally-sensitive fluorescent biosensors to probe the relative abundance, activity and conformational dynamics of CDK/cyclin kinases. CDKSES and CDKACT biosensors have been applied in vitro, in cellulo and in vivo to monitor differences between healthy and cancer cell lines, when tampering with CDK or Cyclin levels using sira, or following treatment with drugs that interfere with kinase activity. CDKCF biosensors have been applied to high throughput screening assays, leading to the successful identification of allosteric modulators of CDK2 kinase activation loop. These fluorescent biosensors constitute potent tools for biomedical applications such as fluorescence-based diagnostics and imaging response to therapeutics, as well as for drug discovery programmes. Related References [1] Morris M.C. (2010) Fluorescent Biosensors of Intracellular Targets: from genetically-encoded reporters to modular polypeptide probes. Cell Biochem.Biophys. 56: [2] Morris, M.C. (2012) Fluorescent Biosensors for Cancer Cell Imaging and Diagnostics. Biosensors and Cancer, CRC press, Ed. V.Preedy and J.Hunter, ISB [3] Van, T... & Morris, M.C. (2013) Fluorescent sensors of protein kinases: from basics to biomedical applications.progr. Mol. Biol. Trans. Science, 113: [4] Morris MC.(2013) Fluorescent Biosensors - Probing Protein Kinase Function in Cancer and Drug Discovery. Biochim Biophys Acta 1834(7): [5] Prével, C., Pellerano, M., Van, T..., Morris, M.C. (2014) Fluorescent Biosensors for High Throughput Screening of Protein Kinase Inhibitors. Biotechnology J. 9,

73 AMPK Epidermal Inhibition Promotes HuR Cytoplasmic Localization Eliciting Post-transcriptional Inflammatory Response in Psoriasis Geneviève Garcin, 1 Isabelle Guiraud, 2 Matthieu Lacroix, 3 Clémence Genthon, 4 Stéphanie Rialle, 4 Jean- Marie Joujoux, 5 Laurent Meunier, 2,6 Thierry Lavabre-Bertrand, 2,7 Pierre-Emmanuel Stoebner, 2,6 and Lionel Le Gallic 2 1 Dynamique des Interactions Membranaires ormales et Pathologiques (DIMP) CRS UMR 5235, Université Montpellier 2, Montpellier 34095, France 2 Laboratoire d Histologie-Embryologie-Cytogénétique Institut des Biomolécules Max Mousseron (IBMM), CRS UMR 5247 Faculté de Médecine Montpellier-îmes, Université Montpellier 1 et 2, îmes 30908, France 3 Institut de Recherche en Cancérologie de Montpellier (IRCM) Institut régional du Cancer Montpellier (ICM), ISERM U896 Université Montpellier 1, Montpellier 34298, France 4 Montpellier Genomix (MGX), Institut de Génomique Fonctionnelle (IGF) CRS ISERM UMS3426, Université Montpellier 1 et 2, Montpellier 34095, France 5 Service d Anatomopathologie, Centre Hospitalier Universitaire de îmes, îmes 30029, France 6 Service de Dermatologie Centre Hospitalier Universitaire de îmes, îmes 30029, France 7 Laboratoire de Cytologie Clinique et Cytogénétique Centre Hospitalier Universitaire de îmes, îmes 30029, France [email protected] IL-20 cytokines areinvolved in the establishment of psoriasis, a common chronic skin inflammation epidemiologically associated with metabolic syndrome, but molecular mechanisms underlying their over-expression remain to be elucidated. We find that keratinocytes (KCs) expressed IL-20 and lymphocytes expressed IL-22 cytokines up-regulation occurs at post-transcriptional level with stabilization of their RA messengers. Looking at psoriatic epidermis, we observethat the p38/mk2 pathway is not activated but that the RA binding protein (RBP) HuR re-localizes in keratinocytes cytoplasm suggesting post-transcriptional regulation of numerous mras. HuRribonucleoproteinimmunoprecipitations analyzed by high throughput sequencing (RIP-Seq) identify potential pre-mature and mature RA targets for uninvolved and involved skin and confirms that HuR activity is displaced from the nucleus to the cytoplasm. umerous psoriasis up-regulated transcripts are HuR targets and HuR knockdownreduce expression of transcripts like bêta-defensin-2, CXCL-10 or IL-20 suggesting an implication of HuR in pathophysiological processes such as morphological, immune and metabolic inflammatory responses. Finally, metabolic disorders affecting psoriatic keratinocytes are responsible of HuR cytoplasmic localization since a decreased activity of the cellular metabolic sensor AMPK, that is observed in human psoriatic epidermis, is sufficient to promote HuR cytosolic localization as well as IL-20 over-production both in human keratinocytes and in vivo in mouse epidermis where it then initiates psoriasis like histological changes. These results provide insights into molecular links between metabolism and post-transcriptional networks during chronic inflammation, as illustrated in psoriasis by mechanisms connecting AMPK, HuR and IL-20.