Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 Leonidas Zervas Award Lectures L1. PEPTIDE-LIPID DUETS IN VIRUSES, BACTERIA, AND HUMANS: NEW VIEWS, NEW AVENUES M. A. R. B. Castanho Instituto de Medicina Molecular, School of Medicine, University of Lisbon 1649-028 Lisbon, Portugal [email protected] There are many biological processes that depend on the interaction between peptides/proteins and membrane lipids, such as viral fusion, translocation across epithelia or innate immune defense. Some of these may be inspiring to develop innovative therapeutic tools. The goal of our group is to unravel the physical principles that govern lipid-peptide interactions, with implications in viral fusion (HIV and Dengue virus are of particular interest), analgesia, antimicrobials, and drug delivery agents. Three hypotheses will be presented and discussed: 1) Viral capsid proteins, usually classified as “structural” (as opposed to “functional”), might have a role in the cell entry mechanism for their membrane-translocating properties. These proteins are rich in domains that are potential Cell Penetrating Peptides (CPP). The example of Dengue virus will be explored as a proof-ofconcept. 2) The action of Antimicrobial Peptides (AMP) in biophysical models, such as lipid vesicles, can be quantitatively correlated to the biological efficacy of these drugs against bacteria. Our work bridges Biophysics and Microbiology. 3) Endogenous peptides can be modified and used as lipophilic drugs able to translocate membranes and epithelia. Following systemic administration these drugs can, for instance, act efficiently at the Central Nervous System (CNS) level. Kyotorphin, an endogenous analgesic and neuroprotective dipeptide, will be
used as example. Kyotorphin derivatives, mainly an Ibuprofen-kyotorphin conjugate, are powerful analgesic and neuroprotective drugs. Acknowledgements/Funding: Fundação para a Ciência e Tecnologia (FCT), Portugal; Marie Curie Actions IAPP and IOF, REA, European Commission, FP7; Ciência Sem Fronteira (Science Without Borders), CAPES and CNPq, Brazil.
M. A. R. B. Castanho, Biography Miguel Castanho is born in 1967 in Santarém, Portugal. Currently he has a Professor position of Biochemistry at the School of Medicine, University of Lisbon. He obtained a Ph.D. degree in Physical Chemistry from Technical University of Lisbon, Portugal. Further, he had several postdoctoral positions at the Faculty of Sciences (University of Lisbon) and Instituto Superior Técnico (Technical University of Lisbon) as well as at the University of Hawaii, Manoa, USA. He became an Assistant Professor at the Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, Portugal in 1994 and an Associate Professor in 2002. He is the co-author of more than 200 peer-reviewed publications. He is a member of the editorial advisory board of several scientific journals. In addition to the scientific achievements mentioned above, he has organized and served as chairman of specialized workshops in the field of Peptide Science, has been invited editor for a special themed issue of the Journal of Peptide Science, serves in the editorial/advisory board of the Journal of Peptide Science and Biopolymers Peptide Science, and is national representative in the Council of the European Peptide Society.
Leonidas Zervas Award Lectures L2. MAKING NEW CONNECTIONS IN PEPTIDE AND PROTEIN CHEMISTRY Philip E. Dawson
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Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 Departments of Chemistry and Cell & Molecular Biology,The Scripps Research Institute, La Jolla, CA 92037 Chemical ligation approaches have become essential tools for the engineering of complex molecules including proteins, nucleic acids and nanoparticles. What makes these reactions so useful is their compatibility with the biological "solvent" water, and a high level of chemoselectivity that enables their application in complex molecular environments. We have worked to develop several ligation chemistries that are highly chemoselective and have sufficient ligation rates to be useful at low concentrations. The optimization of the native chemical ligation methodology, improved routes to the required peptide intermediates, and application of these methods to complex targets will be presented. There is a growing need for side chain specific reactions for the labeling of naturally occurring proteins, we have developed a protein tagging approach that is orthogonal to the more typical maleimide / NHS conjugation reactions.Finally, we have addressed the challenge of covalent assembly of macromolecules and nanoparticles. In these systems, a "native" linkage is irrelevant and the main criteria for a successful ligation methodology are fast reaction rates and high chemoselectivity. We have found that the specific catalysis of imine type reactions enable the controlled assembly and disassembly of macromolecular complexes in aqueous solution at micromolar concentrations.
P. E. Dawson, Biography
Postdoctoral Studies. Philip Dawson is the coauthor of more than 130 peer-reviewed publications as well as numerous book chapters and reviews.
Josef Rudinger Memorial Lecture L3. USE OF PEPTIDES TO MODULATE PROTEIN-PROTEIN INTERACTIONS E. Giralt Institute for Research in Biomedicine, Barcelona, Spain and Department of Organic Chemistry, University of Barcelona, Spain The breakthrough concept that proteins function as a contact network rather than as independent individuals is not only one of the most important advances in our comprehension of living systems, but also translates to a new era in drug discovery. The few reported examples of diseases caused by “impolite” protein social behavior certainly represent only the tip of the iceberg. Therapeutic intervention through molecules designed to selectively modulate the strength and specificity of protein-protein interactions (PPIs) is becoming a reality. This will not only feature molecules with inhibitory capacity: equally or even more interesting are those compounds which can rescue preestablished interactions or structures whose loss results in disease. PPIs are the result of an ensemble of exquisitely regulated molecular recognition events that take place at protein surfaces. Inspection of proteinprotein interfaces allows distinguishing two categories of PPIs: domain-domain and peptidemediated PPIs. Relatively rigid peptides and peptidomimetics have proved to be very efficient inhibitors of this last class of interactions. In this presentation, recent results from our group related to the use of peptides to modulate PPIs will be discussed. This include, among others: the use of all-D linear peptides to modulate beta-amyloid aggregation; the design of cyclic hexapeptide libraries for molecular
Philip E. Dawson holds degrees in Chemistry from Washington University. He obtained a Ph.D. degree in The Scripps Research Institute in 1996. Further, he did postdoctoral research in The California Institute of Technology. He became an Assistant Professor at The Scripps Research Institute in 1997 following by an Associate Professor position in 2005. He is now a member of the Academic Advisory Council and Associate Dean of Graduate and S25
Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 recognition of protein surfaces; and the recent development of cell-permeable photoswitchable PPI inhibitors, that opens the way to manipulating a specific PPI locally and in a time-controlled manner using illumination patterns.
E. Giralt, Biography Ernest Giralt is a Professor of Organic Chemistry in the University of Barcelona, Spain. He obtained his Ph.D. in Chemistry from University of Barcelona, Spain (1974) and undertook postdoctoral studies at the University of Montpellier, France. Ernest Giralt is a member of the editorial advisory board of numerous scientific journals. He is the coauthor of more than 360 peer-reviewed publications as well as several books and book chapters.
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Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 PL1. CHARACTERIZATION OF A NEW PEPTIDE EXHIBITING DOPAMINE EFFECTS BY TARGETTING BRAIN ANGIOTENSIN CONVERTING ENZYME
PL2. WHAT WAS FIRST, THE CODE OR ITS PRODUCTS?
J. Martinez
Department of Structural Biology, Weizmann Institute, Rehovot, Israel
Institut des Biomolécules Max Mousseron UMR 5247, Université de Montpellier, CNRS, ENSCM, Faculté de Pharmacie, 15 Av. C. Flahault, BP 14491, 34093 Montpellier, France We have discovered and characterized a new nonapeptide named “Acein” (JMV3042), which, when injected into the brain of rodents, exhibits dopamine-like effects. This peptide is able to bind to a single class of binding sites with a high affinity in the nanomolar range, specifically localized in the Striatum and Substantia Nigra of rodent brains. Cross-linking experiments showed that the peptide was able to bind a glycosylated protein of about 160 Kda. Using a photoactivable derivative, the striatal protein target of JMV3042 was characterized by a photoaffinity UV cross-linking approach combined with subsequent affinity purification of the ligand covalently bound to its receptor. We succeeded to purify a ~151 kDa protein that was identified by MS/MS as Angiotensin Converting Enzyme (ACE). The activity of JMV3042 on ACE bound membranes in the brain is promising for further investigation of the peptide-dopaminergic system interactions and associated pathologies. This new peptide and its target opens the way to the study of an original dopamine release and regulation pathway, and on the pathologies associated with the deregulation of dopamine balance in the Central Nervous System. Further investigations should focus on the neuroprotective capacity of the action of this peptide and its analogues on brain membrane bound ACE. Key words: Acein, Dopamine, Angiotensin Converting Enzyme.
GENETIC
A. Yonath
Ribosomes, the universal cellular machines for translation of the genetic code into proteins, possess spectacular architecture accompanied by inherent mobility, allowing for their smooth performance as polymerases that translate the genetic code into proteins. The site for peptide bond formation is located within a universal internal semi-symmetrical region. The high conservation of this region implies its existence irrespective of environmental conditions and indicates that it may represent an ancient RNA machine. Hence, it could be the kernel around which life originated. The mechanistic and genetic applications of this finding will be discussed. PL3. INTRODUCING LASSO PEPTIDES AS A MOLECULAR SCAFFOLD FOR DRUG DESIGN M. A. Marahiel Department of Chemistry/Biochemistry Philipps-University Marburg, Hans-MeerweinStrasse 4, D-35043 Marburg, Germany A widespread class of therapeutically important natural products is of peptidic origin. They are either assembled on the ribosome (such as lasso peptides) followed by an extensive posttranslational modification or are produced independent of the ribosome (non-ribosomally) using large multi-modular-enzymes, the so called Non-Ribosomal Peptide Synthetases (NRPS)[1]. One class of macrocyclic peptides we are studying is of ribosomal origin. These bioactive
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Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 natural products synthesized by bacteria are composed of 16-21 canonical amino acids and show unusual and complex lasso-structures [2]. They share an N-terminal macrolactam ring comprising 7 to 9 residue, generated upon leader peptide splitting and the condensation reaction between the generated α-NH2 group of an N-terminal residue and the carboxyl side chain of Asp/Glu at position 7, 8 or 9 [3]. The tail (8 - 13 residues) threads through the macrocycle and is trapped by steric hindrance of bulky side chains within the ring, generating an uncommon lariat knot structure of unmatched high stability. Their biological activities range from inhibition of HIV replication to blockage of the bacterial RNA polymerase. In this superfamily of lasso-structure peptides, we are interested in studying their NMR structures [4], enzymatic biosynthesis [5] and in developing methods for their reengineering as a molecular scaffold for drug design [6]. Key words: Peptide antibiotics, Lasso peptides, NMR Structure, peptide grafting [1] [2] [3]
[4] [5]
[6]
M.A. Marahiel, J. Pept. Sci. 15, 799 (2009) J.D. Hegemann, M. Zimmermann, S. Zhu, D. Klug, and M.A. Marahiel, Biopolymers, 100, 527 (2013) J.D. Hegemann, M. Zimmermann, S. Zhu, H. Steuber, K. Harms, X. Xie, and M.A. Marahiel, Angew. Chem., 126, 2230 (2014) X. Xie and M.A. Marahiel, ChemBiochem13, 621 (2012) K.P. Yan, Y. Li, S. Zirah, C. Goulard, T.A. Knappe, M. A. Marahiel, and S. Rebuffat, ChemBiochem 13, 1046 (2012) T.A. Knappe, F., Manzenrieder, C., Mas-Moruno, U. Linne, F. Sasse, H. Kessler, X. Xie, and M.A. Marahiel, Angew. Chem. Int. Ed. Engl. 50, 8714 (2011)
PL4. MOLECULAR MECHANISM PEPTIDE HORMONE BINDING GPCR TRAFFICKING
OF AND
A. G. Beck-Sickinger, S. Els-Heindl, K. Kostelnik, V. Maede, S. Babilon, X. Pedragosa-Badia, K. Moerl University of Leipzig, Institute of Biochemistry, Bruederstr. 34, 04103 Leipzig
G-protein couple receptors (GPCR) are an important class of targets for drug development [1]. Many peptides including ghrelin, pancreatic polypeptide and neuropeptide Y transmit their biological activity by GPCR. In order to develop peptide drugs, however, several prerequisites are required including high and selective interaction with the receptor [2], agonism or inverse agonism according to the desired function [3] as well as improved proteolytic stability and bioavailability. As many GPCR are prone of arrestin dependent internalization after agonist binding this has to be considered as well for drug development [4]. Here we describe novel approaches to rationally determine peptide modification after molecular knowledge of their binding mode [5]. Furthermore approaches to identify peptide degradation in vivo will be presented. Finally, modifications that direct trafficking have been identified and shown to influence receptor localization either on the surface or to induce internalization [6]. This is of major relevance for all types of peptide drugs. Examples will be shown from the field of anorexic peptides targeting the Y2- or the Y4-receptors as well as from the orexigenic ghrelin receptor. Key words: G protein coupled receptors, peptide therapeutics, trafficking, chemical modification, neuropeptide Y, ghrelin [1] [2] [3] [4] [5] [6]
A. V. Ahrens et al. Future Med Chem., 4, 1567 (2012) X. Pedragosa-Badia et al. Front Endocrinol (Lausanne), 4, 5 (2013) S. Els et al. J. Med. Chem., 55, 7437 (2012) S. Babilon et al., Biol. Chem., 394, 921(2013) X. Pedragosa-Badia et al. J. Biol. Chem., 289, 5846 (2014) V. Maede et al. Angew. Chem. Int. Ed., accepted (2014)
PL5. DEVELOPING STABLE, BIOACTIVE, NON-TOXIC PEPTIDES AND PEPTIDOMIMETICS AS DRUGS FOR DISEASE Victor J. Hruby
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Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, U.S.A. Most of our current drugs for degenerative diseases such as diabetes, prolonged and neuropathic pain, cancer, cardiovascular disease, etc. treat symptoms of the disease and have toxicities. Peptides and peptidomimetics have great potential for the development of novel drugs that can treat the disease state, with high potency and efficacy, and have greatly reduced or none of the toxicities of current drugs. The peptide/protein scaffold is the nontoxic biological scaffold and offers unique opportunities for development of drugs. There recently has been an increased awareness in the medical and drug design communities of their potential. We will discuss strategies that can be used to develop peptides and peptidomimetic ligands that are more stable, have enhanced bioavailability, can cross membrane barriers and have minimal or no toxicities. Specific examples will include novel multivalent ligands for the treatment of prolonged and neuropathic pain without the development of tolerance, addiction and toxicities of current drugs for pain. Additional examples of multivalent ligands for detection and treatment of cancer, and of highly potent, selective and bioavailable ligands for the melanocortin receptors with applications to cancer, pigmentary disorders, inflammation, pain and other disorders will be discussed as time permits. Special emphasis to the conformational and structural considerations critical to these developments will be presented. Acknowledgements: Supported in part by grants from the U.S. Public Health Service, National Institutes of Health, NIDA, NCI and GM Key Words: peptide drug design, multivalent ligands, drugs for disease state, stable peptides
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Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 IO1. SUNFLOWER TRYPSIN INHIBITOR-1 (SFTI-1) AS USEFUL SCAFFOLD FOR THE DEVELOPMENT OF PEPTIDEBASED DRUG LEADS K. Rolka Faculty of Chemistry, University of Gdansk, Poland
diagnostic agents. In this presentation, examples of possible applications of peptides based on SFTI-1 structure will be discussed. Key words: SFTI-1, proteinase, inhibitors, peptide splicing, cell-penetrating
IO2. DELINEATING THE FUNCTION OF AN SECRETASE-DERIVED PRION PEPTIDE UNRAVELS A MOLECULAR CROSSTALK BETWEEN CELLULAR PRION AND ALZHEIMER’S DISEASE PATHOLOGY
SFTI-1, the trypsin inhibitor isolated in 1999 from the seeds of sunflower, is currently the smallest circular peptide consisting of proteinogenic amino acids derived from natural sources. Its circular backbone that comprises F. Checler and M-V. Guillot-Sestier 14 amino acid residues is additionally constrained by the disulfide bridge. Lys5 IPMC, UMR7275 CNRS-UNS, Valbonne, located in the substrate specificity P1 position is France responsible for its trypsin-like inhibitory established that the cellular activity. Owing to its exceptionally small size, We have previously c compact structure, and high inhibitory activity, prion PrP undergoes constitutive and regulated SFTI-1 has become a very attractive template cleavages by disintegrins ADAM10 and for designing proteinase inhibitors against ADAM17 [1], yielding a secreted fragment N1 a membrane-attached C-terminal physiologically important enzymes. In addition, and several groups, including ours, showed that counterpart C1. This cleavage is reminiscent of fluorescent labeled SFTI-1 and its linear the one that takes place on the -amyloid backbone analogues are able topenetrate cells. precursor protein APP. We have shown that In the case of analogues unmodified in P1 N1 is a protective fragment that displays its position, such analogues retain inhibitory antiapoptotic function via the down regulation activity and proteolytic stability. Interestingly, of the tumor suppressor p53, both in cells and in appropriately designed analogues, when vivo [2]. Recently, we have shown that N1 also incubated with cognate enzymes undergo protects cells against the toxicity harbored by proteolytic process resulting in cutting out a A oligomers that accumulate in Azheimer’s middle fragment of the peptide and resynthesis disease pathology. We discuss the possibility of the new peptide bond. Such phenomenon that -secretase-mediated formation of N1 resembles peptide splicing previously reported could correspond to a protective cellular for proteasomes, significantly more complex pathway taking place as a compensatory proteinases. One can speculate that protease- mechanism at early asymptomatic phase of catalyzed peptide splicing is another mechanism Alzheimer’s disease. This study delineates bridges linking distinct that increases the diversity of biologically active molecular peptides. On the other hand, such a system may neurodegenerative pathologies. be utilized for introduction of site-selective [1] VINCENT, B., PAITEL, E., SAFTIG, P., FROBERT, synthetic moieties as inserts into spliced Y., DE STROOPER, B., GRASSI, J., BLACK, R., peptides. Taking into the consideration that LOPEZ- PEREZ, E., CHECLER, F. J. Biol. Chem. SFTI-1 can cross cell membranes, such 276, 37743 (2001) analogues may serve as therapeutic or S30
Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 [2]
[3]
GUILLOT-SESTIER, M-V., SUNYACH, C., DRUON, C., SCARZELLO, S. and CHECLER, F.J. Biol. Chem. 284, 35973 (2009) GUILLOT-SESTIER, M.V., SUNYACH, C., FERREIRA, S.T, MARZOLO, M-P., BAUER, C., THEVENET, A.,CHECLER, F. (2012) J. Biol. Chem. 287, 5021-5032.
IO3. MULTIFUNCTIONALITY OF THE SEQUENTIAL OLIGOPEPTIDE CARRIERS (SOC). ANTIGENICITY, IMMUNOGENICITY, CELL PENETRATING AND ANTITUMOR ACTIVITY V. Tsikaris, S. Papas, C. Papadopoulos, C. Sakarellos, M. Daitsiotis-Sakarellos, D. Krikorian, A. Tzakos, E. Giannopoulou , H. Kalofonos
A diverse array of carriers has appeared in the literature such as proteins, polysaccharides and liposomes [1]. In this study we will present the multifunctionality of the Sequential Oligopeptide Carriers (SOC) formed by the repetitive (Lys-Aib-Cys) [2] or (Lys-Aib-Gly) [3] triads. Those carriers are capable of conjugating B and T cell epitopesto obtain potent antigens or immunogens [4] and to penetrate into cells. Moreover a class of them showed significant antitumor activity. Key words: biocargo, disulfide bond, redox sensitive carriers. [1] [2]
[3]
Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of Ioannina, Ioannina, 45110, Greece Advances in genomics, biotechnology and chemistry opened new therapeutic and diagnostic approaches that target intracellular molecules. This progress has proved a major challenge for research scientists and pharmaceutical industry. The key challenge in this area is the development of therapeutic agents that act only where needed. However, the cell membrane penetrating barriers and the lack of cell selectivity of the intracellular acting therapeutic agents limit actually their therapeutic value. Novel therapeutic approaches such as gene and protein therapy have met limited applications due to the cellimpermeable nature of bioactive peptides and oligonucleotides. In addition the selective delivery of bioactive molecules to their site of action which should increase their effectiveness and limit possible side effects is a very complex process. Many efforts have been made to increase the specificity and the successful delivery of biomolecules through bioconjugation of the biomolecule to a carrier.
[4]
R.C. Mustata, A. Grigorescu, S.M. Petrescu, J. Cell Mol. Med., 13, 3110, (2009) S. Papas, T. Akoumianaki, C. Kalogiros, L. Hadjiarapoglou, P. Theodoropoulos, V. Tsikaris. J.Pept. Sci., 13, 662, (2007) V. Tsikaris, C. Sakarellos, M. Sakarellos-Daitsiotis, P. Orlewski, M. Marraud, M.T. Cung, E. Vatzaki, S. Tzartos, Int. J. Biol. Macromol,19, 195,(1996) M. Sakarellos-Daitsiotis, C. Alexopoulos, C. Sakarellos, J. Pharm. Biomed. Anal., 34, 761, (2004)
IO4. CHEMICAL UBIQUITINATION H. Ovaa Division of Cell Biology, the Netherlands Cancer Institute, Amsterdam Although 8 ubiquitin chain topoisomers are found in nature, we can enzymatically generate and study only few of them. The same holds true for ubiquitinated or ubiquitin-like modified polypeptides. Only for few polypeptide sequences and proteins the ligase combinations are known and can be isolated, needed for ubiquitin or ubiquitin-like modification in vitro. We have deveoped chemical strategies that allow the construction of virtually any ubiquitin or ubiquitin-like conjugate. This technology has made custom ubiquitination services reality and the basics of this technology will be explainedin detail. In addition, a series of novel ubiquitin-based probes will be discussed that have been
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Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 designed to study the activity of deubiquitinating enzymes (DUBs). These probes are based on the ubiquitin structure and are equipped with a reactive moiety that allows covalent inhibition of DUBs through reaction with their active site cysteine nucleophiles. This strategy allows for example the identification of novel DUBs and the simultaneous activity measurement of multiple cysteine-dependent DUBs present in a given example. We recently discovered by serendipity DUB probes based on alkynes as a reactive moiety. Interestingly, these alkynes have so far been considered to be unreactive but it appears that DUBs can trigger a chemical reaction not yet known. The discovery and implications of this unexpected alkyne reactivity will be discussed. Alkyne-based DUB probes react with a wider range of DUBs compared to other probes
previously developed while they seem inert to reactions other than reactions with active site cysteine residues in deubiquitinating enzymes.
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used as example. Kyotorphin derivatives, mainly an Ibuprofen-kyotorphin conjugate, are powerful analgesic and neuroprotective drugs. Acknowledgements/Funding: Fundação para a Ciência e Tecnologia (FCT), Portugal; Marie Curie Actions IAPP and IOF, REA, European Commission, FP7; Ciência Sem Fronteira (Science Without Borders), CAPES and CNPq, Brazil.
M. A. R. B. Castanho, Biography Miguel Castanho is born in 1967 in Santarém, Portugal. Currently he has a Professor position of Biochemistry at the School of Medicine, University of Lisbon. He obtained a Ph.D. degree in Physical Chemistry from Technical University of Lisbon, Portugal. Further, he had several postdoctoral positions at the Faculty of Sciences (University of Lisbon) and Instituto Superior Técnico (Technical University of Lisbon) as well as at the University of Hawaii, Manoa, USA. He became an Assistant Professor at the Department of Chemistry and Biochemistry, Faculty of Sciences, University of Lisbon, Portugal in 1994 and an Associate Professor in 2002. He is the co-author of more than 200 peer-reviewed publications. He is a member of the editorial advisory board of several scientific journals. In addition to the scientific achievements mentioned above, he has organized and served as chairman of specialized workshops in the field of Peptide Science, has been invited editor for a special themed issue of the Journal of Peptide Science, serves in the editorial/advisory board of the Journal of Peptide Science and Biopolymers Peptide Science, and is national representative in the Council of the European Peptide Society.
Leonidas Zervas Award Lectures L2. MAKING NEW CONNECTIONS IN PEPTIDE AND PROTEIN CHEMISTRY Philip E. Dawson
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Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 Departments of Chemistry and Cell & Molecular Biology,The Scripps Research Institute, La Jolla, CA 92037 Chemical ligation approaches have become essential tools for the engineering of complex molecules including proteins, nucleic acids and nanoparticles. What makes these reactions so useful is their compatibility with the biological "solvent" water, and a high level of chemoselectivity that enables their application in complex molecular environments. We have worked to develop several ligation chemistries that are highly chemoselective and have sufficient ligation rates to be useful at low concentrations. The optimization of the native chemical ligation methodology, improved routes to the required peptide intermediates, and application of these methods to complex targets will be presented. There is a growing need for side chain specific reactions for the labeling of naturally occurring proteins, we have developed a protein tagging approach that is orthogonal to the more typical maleimide / NHS conjugation reactions.Finally, we have addressed the challenge of covalent assembly of macromolecules and nanoparticles. In these systems, a "native" linkage is irrelevant and the main criteria for a successful ligation methodology are fast reaction rates and high chemoselectivity. We have found that the specific catalysis of imine type reactions enable the controlled assembly and disassembly of macromolecular complexes in aqueous solution at micromolar concentrations.
P. E. Dawson, Biography
Postdoctoral Studies. Philip Dawson is the coauthor of more than 130 peer-reviewed publications as well as numerous book chapters and reviews.
Josef Rudinger Memorial Lecture L3. USE OF PEPTIDES TO MODULATE PROTEIN-PROTEIN INTERACTIONS E. Giralt Institute for Research in Biomedicine, Barcelona, Spain and Department of Organic Chemistry, University of Barcelona, Spain The breakthrough concept that proteins function as a contact network rather than as independent individuals is not only one of the most important advances in our comprehension of living systems, but also translates to a new era in drug discovery. The few reported examples of diseases caused by “impolite” protein social behavior certainly represent only the tip of the iceberg. Therapeutic intervention through molecules designed to selectively modulate the strength and specificity of protein-protein interactions (PPIs) is becoming a reality. This will not only feature molecules with inhibitory capacity: equally or even more interesting are those compounds which can rescue preestablished interactions or structures whose loss results in disease. PPIs are the result of an ensemble of exquisitely regulated molecular recognition events that take place at protein surfaces. Inspection of proteinprotein interfaces allows distinguishing two categories of PPIs: domain-domain and peptidemediated PPIs. Relatively rigid peptides and peptidomimetics have proved to be very efficient inhibitors of this last class of interactions. In this presentation, recent results from our group related to the use of peptides to modulate PPIs will be discussed. This include, among others: the use of all-D linear peptides to modulate beta-amyloid aggregation; the design of cyclic hexapeptide libraries for molecular
Philip E. Dawson holds degrees in Chemistry from Washington University. He obtained a Ph.D. degree in The Scripps Research Institute in 1996. Further, he did postdoctoral research in The California Institute of Technology. He became an Assistant Professor at The Scripps Research Institute in 1997 following by an Associate Professor position in 2005. He is now a member of the Academic Advisory Council and Associate Dean of Graduate and S25
Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 recognition of protein surfaces; and the recent development of cell-permeable photoswitchable PPI inhibitors, that opens the way to manipulating a specific PPI locally and in a time-controlled manner using illumination patterns.
E. Giralt, Biography Ernest Giralt is a Professor of Organic Chemistry in the University of Barcelona, Spain. He obtained his Ph.D. in Chemistry from University of Barcelona, Spain (1974) and undertook postdoctoral studies at the University of Montpellier, France. Ernest Giralt is a member of the editorial advisory board of numerous scientific journals. He is the coauthor of more than 360 peer-reviewed publications as well as several books and book chapters.
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Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 PL1. CHARACTERIZATION OF A NEW PEPTIDE EXHIBITING DOPAMINE EFFECTS BY TARGETTING BRAIN ANGIOTENSIN CONVERTING ENZYME
PL2. WHAT WAS FIRST, THE CODE OR ITS PRODUCTS?
J. Martinez
Department of Structural Biology, Weizmann Institute, Rehovot, Israel
Institut des Biomolécules Max Mousseron UMR 5247, Université de Montpellier, CNRS, ENSCM, Faculté de Pharmacie, 15 Av. C. Flahault, BP 14491, 34093 Montpellier, France We have discovered and characterized a new nonapeptide named “Acein” (JMV3042), which, when injected into the brain of rodents, exhibits dopamine-like effects. This peptide is able to bind to a single class of binding sites with a high affinity in the nanomolar range, specifically localized in the Striatum and Substantia Nigra of rodent brains. Cross-linking experiments showed that the peptide was able to bind a glycosylated protein of about 160 Kda. Using a photoactivable derivative, the striatal protein target of JMV3042 was characterized by a photoaffinity UV cross-linking approach combined with subsequent affinity purification of the ligand covalently bound to its receptor. We succeeded to purify a ~151 kDa protein that was identified by MS/MS as Angiotensin Converting Enzyme (ACE). The activity of JMV3042 on ACE bound membranes in the brain is promising for further investigation of the peptide-dopaminergic system interactions and associated pathologies. This new peptide and its target opens the way to the study of an original dopamine release and regulation pathway, and on the pathologies associated with the deregulation of dopamine balance in the Central Nervous System. Further investigations should focus on the neuroprotective capacity of the action of this peptide and its analogues on brain membrane bound ACE. Key words: Acein, Dopamine, Angiotensin Converting Enzyme.
GENETIC
A. Yonath
Ribosomes, the universal cellular machines for translation of the genetic code into proteins, possess spectacular architecture accompanied by inherent mobility, allowing for their smooth performance as polymerases that translate the genetic code into proteins. The site for peptide bond formation is located within a universal internal semi-symmetrical region. The high conservation of this region implies its existence irrespective of environmental conditions and indicates that it may represent an ancient RNA machine. Hence, it could be the kernel around which life originated. The mechanistic and genetic applications of this finding will be discussed. PL3. INTRODUCING LASSO PEPTIDES AS A MOLECULAR SCAFFOLD FOR DRUG DESIGN M. A. Marahiel Department of Chemistry/Biochemistry Philipps-University Marburg, Hans-MeerweinStrasse 4, D-35043 Marburg, Germany A widespread class of therapeutically important natural products is of peptidic origin. They are either assembled on the ribosome (such as lasso peptides) followed by an extensive posttranslational modification or are produced independent of the ribosome (non-ribosomally) using large multi-modular-enzymes, the so called Non-Ribosomal Peptide Synthetases (NRPS)[1]. One class of macrocyclic peptides we are studying is of ribosomal origin. These bioactive
S27
Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 natural products synthesized by bacteria are composed of 16-21 canonical amino acids and show unusual and complex lasso-structures [2]. They share an N-terminal macrolactam ring comprising 7 to 9 residue, generated upon leader peptide splitting and the condensation reaction between the generated α-NH2 group of an N-terminal residue and the carboxyl side chain of Asp/Glu at position 7, 8 or 9 [3]. The tail (8 - 13 residues) threads through the macrocycle and is trapped by steric hindrance of bulky side chains within the ring, generating an uncommon lariat knot structure of unmatched high stability. Their biological activities range from inhibition of HIV replication to blockage of the bacterial RNA polymerase. In this superfamily of lasso-structure peptides, we are interested in studying their NMR structures [4], enzymatic biosynthesis [5] and in developing methods for their reengineering as a molecular scaffold for drug design [6]. Key words: Peptide antibiotics, Lasso peptides, NMR Structure, peptide grafting [1] [2] [3]
[4] [5]
[6]
M.A. Marahiel, J. Pept. Sci. 15, 799 (2009) J.D. Hegemann, M. Zimmermann, S. Zhu, D. Klug, and M.A. Marahiel, Biopolymers, 100, 527 (2013) J.D. Hegemann, M. Zimmermann, S. Zhu, H. Steuber, K. Harms, X. Xie, and M.A. Marahiel, Angew. Chem., 126, 2230 (2014) X. Xie and M.A. Marahiel, ChemBiochem13, 621 (2012) K.P. Yan, Y. Li, S. Zirah, C. Goulard, T.A. Knappe, M. A. Marahiel, and S. Rebuffat, ChemBiochem 13, 1046 (2012) T.A. Knappe, F., Manzenrieder, C., Mas-Moruno, U. Linne, F. Sasse, H. Kessler, X. Xie, and M.A. Marahiel, Angew. Chem. Int. Ed. Engl. 50, 8714 (2011)
PL4. MOLECULAR MECHANISM PEPTIDE HORMONE BINDING GPCR TRAFFICKING
OF AND
A. G. Beck-Sickinger, S. Els-Heindl, K. Kostelnik, V. Maede, S. Babilon, X. Pedragosa-Badia, K. Moerl University of Leipzig, Institute of Biochemistry, Bruederstr. 34, 04103 Leipzig
G-protein couple receptors (GPCR) are an important class of targets for drug development [1]. Many peptides including ghrelin, pancreatic polypeptide and neuropeptide Y transmit their biological activity by GPCR. In order to develop peptide drugs, however, several prerequisites are required including high and selective interaction with the receptor [2], agonism or inverse agonism according to the desired function [3] as well as improved proteolytic stability and bioavailability. As many GPCR are prone of arrestin dependent internalization after agonist binding this has to be considered as well for drug development [4]. Here we describe novel approaches to rationally determine peptide modification after molecular knowledge of their binding mode [5]. Furthermore approaches to identify peptide degradation in vivo will be presented. Finally, modifications that direct trafficking have been identified and shown to influence receptor localization either on the surface or to induce internalization [6]. This is of major relevance for all types of peptide drugs. Examples will be shown from the field of anorexic peptides targeting the Y2- or the Y4-receptors as well as from the orexigenic ghrelin receptor. Key words: G protein coupled receptors, peptide therapeutics, trafficking, chemical modification, neuropeptide Y, ghrelin [1] [2] [3] [4] [5] [6]
A. V. Ahrens et al. Future Med Chem., 4, 1567 (2012) X. Pedragosa-Badia et al. Front Endocrinol (Lausanne), 4, 5 (2013) S. Els et al. J. Med. Chem., 55, 7437 (2012) S. Babilon et al., Biol. Chem., 394, 921(2013) X. Pedragosa-Badia et al. J. Biol. Chem., 289, 5846 (2014) V. Maede et al. Angew. Chem. Int. Ed., accepted (2014)
PL5. DEVELOPING STABLE, BIOACTIVE, NON-TOXIC PEPTIDES AND PEPTIDOMIMETICS AS DRUGS FOR DISEASE Victor J. Hruby
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Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 Department of Chemistry and Biochemistry, University of Arizona, Tucson, Arizona 85721, U.S.A. Most of our current drugs for degenerative diseases such as diabetes, prolonged and neuropathic pain, cancer, cardiovascular disease, etc. treat symptoms of the disease and have toxicities. Peptides and peptidomimetics have great potential for the development of novel drugs that can treat the disease state, with high potency and efficacy, and have greatly reduced or none of the toxicities of current drugs. The peptide/protein scaffold is the nontoxic biological scaffold and offers unique opportunities for development of drugs. There recently has been an increased awareness in the medical and drug design communities of their potential. We will discuss strategies that can be used to develop peptides and peptidomimetic ligands that are more stable, have enhanced bioavailability, can cross membrane barriers and have minimal or no toxicities. Specific examples will include novel multivalent ligands for the treatment of prolonged and neuropathic pain without the development of tolerance, addiction and toxicities of current drugs for pain. Additional examples of multivalent ligands for detection and treatment of cancer, and of highly potent, selective and bioavailable ligands for the melanocortin receptors with applications to cancer, pigmentary disorders, inflammation, pain and other disorders will be discussed as time permits. Special emphasis to the conformational and structural considerations critical to these developments will be presented. Acknowledgements: Supported in part by grants from the U.S. Public Health Service, National Institutes of Health, NIDA, NCI and GM Key Words: peptide drug design, multivalent ligands, drugs for disease state, stable peptides
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Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 IO1. SUNFLOWER TRYPSIN INHIBITOR-1 (SFTI-1) AS USEFUL SCAFFOLD FOR THE DEVELOPMENT OF PEPTIDEBASED DRUG LEADS K. Rolka Faculty of Chemistry, University of Gdansk, Poland
diagnostic agents. In this presentation, examples of possible applications of peptides based on SFTI-1 structure will be discussed. Key words: SFTI-1, proteinase, inhibitors, peptide splicing, cell-penetrating
IO2. DELINEATING THE FUNCTION OF AN SECRETASE-DERIVED PRION PEPTIDE UNRAVELS A MOLECULAR CROSSTALK BETWEEN CELLULAR PRION AND ALZHEIMER’S DISEASE PATHOLOGY
SFTI-1, the trypsin inhibitor isolated in 1999 from the seeds of sunflower, is currently the smallest circular peptide consisting of proteinogenic amino acids derived from natural sources. Its circular backbone that comprises F. Checler and M-V. Guillot-Sestier 14 amino acid residues is additionally constrained by the disulfide bridge. Lys5 IPMC, UMR7275 CNRS-UNS, Valbonne, located in the substrate specificity P1 position is France responsible for its trypsin-like inhibitory established that the cellular activity. Owing to its exceptionally small size, We have previously c compact structure, and high inhibitory activity, prion PrP undergoes constitutive and regulated SFTI-1 has become a very attractive template cleavages by disintegrins ADAM10 and for designing proteinase inhibitors against ADAM17 [1], yielding a secreted fragment N1 a membrane-attached C-terminal physiologically important enzymes. In addition, and several groups, including ours, showed that counterpart C1. This cleavage is reminiscent of fluorescent labeled SFTI-1 and its linear the one that takes place on the -amyloid backbone analogues are able topenetrate cells. precursor protein APP. We have shown that In the case of analogues unmodified in P1 N1 is a protective fragment that displays its position, such analogues retain inhibitory antiapoptotic function via the down regulation activity and proteolytic stability. Interestingly, of the tumor suppressor p53, both in cells and in appropriately designed analogues, when vivo [2]. Recently, we have shown that N1 also incubated with cognate enzymes undergo protects cells against the toxicity harbored by proteolytic process resulting in cutting out a A oligomers that accumulate in Azheimer’s middle fragment of the peptide and resynthesis disease pathology. We discuss the possibility of the new peptide bond. Such phenomenon that -secretase-mediated formation of N1 resembles peptide splicing previously reported could correspond to a protective cellular for proteasomes, significantly more complex pathway taking place as a compensatory proteinases. One can speculate that protease- mechanism at early asymptomatic phase of catalyzed peptide splicing is another mechanism Alzheimer’s disease. This study delineates bridges linking distinct that increases the diversity of biologically active molecular peptides. On the other hand, such a system may neurodegenerative pathologies. be utilized for introduction of site-selective [1] VINCENT, B., PAITEL, E., SAFTIG, P., FROBERT, synthetic moieties as inserts into spliced Y., DE STROOPER, B., GRASSI, J., BLACK, R., peptides. Taking into the consideration that LOPEZ- PEREZ, E., CHECLER, F. J. Biol. Chem. SFTI-1 can cross cell membranes, such 276, 37743 (2001) analogues may serve as therapeutic or S30
Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 [2]
[3]
GUILLOT-SESTIER, M-V., SUNYACH, C., DRUON, C., SCARZELLO, S. and CHECLER, F.J. Biol. Chem. 284, 35973 (2009) GUILLOT-SESTIER, M.V., SUNYACH, C., FERREIRA, S.T, MARZOLO, M-P., BAUER, C., THEVENET, A.,CHECLER, F. (2012) J. Biol. Chem. 287, 5021-5032.
IO3. MULTIFUNCTIONALITY OF THE SEQUENTIAL OLIGOPEPTIDE CARRIERS (SOC). ANTIGENICITY, IMMUNOGENICITY, CELL PENETRATING AND ANTITUMOR ACTIVITY V. Tsikaris, S. Papas, C. Papadopoulos, C. Sakarellos, M. Daitsiotis-Sakarellos, D. Krikorian, A. Tzakos, E. Giannopoulou , H. Kalofonos
A diverse array of carriers has appeared in the literature such as proteins, polysaccharides and liposomes [1]. In this study we will present the multifunctionality of the Sequential Oligopeptide Carriers (SOC) formed by the repetitive (Lys-Aib-Cys) [2] or (Lys-Aib-Gly) [3] triads. Those carriers are capable of conjugating B and T cell epitopesto obtain potent antigens or immunogens [4] and to penetrate into cells. Moreover a class of them showed significant antitumor activity. Key words: biocargo, disulfide bond, redox sensitive carriers. [1] [2]
[3]
Department of Chemistry, Section of Organic Chemistry and Biochemistry, University of Ioannina, Ioannina, 45110, Greece Advances in genomics, biotechnology and chemistry opened new therapeutic and diagnostic approaches that target intracellular molecules. This progress has proved a major challenge for research scientists and pharmaceutical industry. The key challenge in this area is the development of therapeutic agents that act only where needed. However, the cell membrane penetrating barriers and the lack of cell selectivity of the intracellular acting therapeutic agents limit actually their therapeutic value. Novel therapeutic approaches such as gene and protein therapy have met limited applications due to the cellimpermeable nature of bioactive peptides and oligonucleotides. In addition the selective delivery of bioactive molecules to their site of action which should increase their effectiveness and limit possible side effects is a very complex process. Many efforts have been made to increase the specificity and the successful delivery of biomolecules through bioconjugation of the biomolecule to a carrier.
[4]
R.C. Mustata, A. Grigorescu, S.M. Petrescu, J. Cell Mol. Med., 13, 3110, (2009) S. Papas, T. Akoumianaki, C. Kalogiros, L. Hadjiarapoglou, P. Theodoropoulos, V. Tsikaris. J.Pept. Sci., 13, 662, (2007) V. Tsikaris, C. Sakarellos, M. Sakarellos-Daitsiotis, P. Orlewski, M. Marraud, M.T. Cung, E. Vatzaki, S. Tzartos, Int. J. Biol. Macromol,19, 195,(1996) M. Sakarellos-Daitsiotis, C. Alexopoulos, C. Sakarellos, J. Pharm. Biomed. Anal., 34, 761, (2004)
IO4. CHEMICAL UBIQUITINATION H. Ovaa Division of Cell Biology, the Netherlands Cancer Institute, Amsterdam Although 8 ubiquitin chain topoisomers are found in nature, we can enzymatically generate and study only few of them. The same holds true for ubiquitinated or ubiquitin-like modified polypeptides. Only for few polypeptide sequences and proteins the ligase combinations are known and can be isolated, needed for ubiquitin or ubiquitin-like modification in vitro. We have deveoped chemical strategies that allow the construction of virtually any ubiquitin or ubiquitin-like conjugate. This technology has made custom ubiquitination services reality and the basics of this technology will be explainedin detail. In addition, a series of novel ubiquitin-based probes will be discussed that have been
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Award Lectures, Plenary Lectures PL 1-5, Invited Lectures IL 1-4 designed to study the activity of deubiquitinating enzymes (DUBs). These probes are based on the ubiquitin structure and are equipped with a reactive moiety that allows covalent inhibition of DUBs through reaction with their active site cysteine nucleophiles. This strategy allows for example the identification of novel DUBs and the simultaneous activity measurement of multiple cysteine-dependent DUBs present in a given example. We recently discovered by serendipity DUB probes based on alkynes as a reactive moiety. Interestingly, these alkynes have so far been considered to be unreactive but it appears that DUBs can trigger a chemical reaction not yet known. The discovery and implications of this unexpected alkyne reactivity will be discussed. Alkyne-based DUB probes react with a wider range of DUBs compared to other probes
previously developed while they seem inert to reactions other than reactions with active site cysteine residues in deubiquitinating enzymes.
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