|Director : Bost Pierre-Etienne (firstname.lastname@example.org)|
The Organic Chemistry Unit deals with the synthesis and the evaluation of biologically active molecules. The design of original products (synthetic vaccines, enzyme inhibitors, etc.) is performed in collaboration with other research units in order to study their physical properties and activities in various biological systems. The ultimate objective includes the validation of new targets and new therapeutic approaches of infectious diseases and cancer. Moreover, a programme of in vivo selection and in vitro directed evolution of enzymes is developed.
Nucleoside monophosphate kinase of Mycobacterium tuberculosis
(Laurence Dugué, Cécile Gasse, Sylvie Pochet and Hélène Munier-Lehmann)
The nucleoside monophosphate kinases (NMPKs) from M. tuberculosis were selected as potential targets for antituberculosis therapy. Two NMPKs, UMPK and TMPK, have been particularly studied. This year the search for TMPK inhibitors was pursued. This work is the subject of a Transversal Research Programm joining different laboratories of the Pasteur Institute.
Inhibitors were designed based on the available 3D structure of TMPK (in collaboration with G. Labesse, D. Douguet). Starting from the non nucleosidic compounds previously synthesized and tested, or from in silico screening of chemical libraries, structurally related molecules have been synthesized (in collaboration with the group of P. Herdewijn and S. Van Calenbergh). Those having micro-molar inhibitory activity have been evaluated on M. tuberculosis cultures. However no growth inhibition has been detected. In order to improve the inhibitory potency, co-crystallisation assays are in progress.
Directed enzyme evolution using selection
(Sophie Vichier-Guerre, Pierre-Alexandre Kaminski and Jean-Luc Jestin)
To design tailor-made catalysts, directed enzyme evolution appears as one of the most promising approaches. Wheras classical directed enzyme evolution strategies make use of screening, we develop selections to analyse simultaneously the catalytic activity of more than 108 distinct proteins. The in vitro selections for catalysis are set up in vivo using appropriate Escherichia coli strains and in vitro using a chemistry of filamentous phages. In 2003, thermostable DNA polymerases endowed with new catalytic activities have been obtained and variant N-deoxyribosyltransferases active on nucleoside analogs have been isolated by in vivo selection. Therapeutic applications of directed enzyme evolution strategies define current projects.
Apoptosis and new therapetic approaches based on PP1/PP2A protein phosphatases
(Alphonse Garcia, Julien Guergnon, Véronique Hospital, Frédéric Dessauge and Victoria Dominguez)
The ser/thr protein phosphatases, type 1 (PP1) and type 2A (PP2A) are evolutionarily conserved enzymes that represent a major portion of serine/threonine phosphatase activity in cell extracts. PP1 and PP2A are holoenzymes composed of oligomeric complexes comprising a catalytic subunit (PP1c) or a core complex (PP2A-A/PR65 structural and PP2Ac catalytic subunit) associated with other interacting proteins corresponding to targeting and regulatory subunits.
Recent results suggest a pivotal role of PP1/PP2A in the regulation of Bcl-2 proteins and cell survival, and lead to a new concept called "Drug Phosphatase Technology" (DPT). This concept that aims to deliver ex vivo and in vivo peptides with sequence surrounding PP1/PP2A binding sites identified in important regulatory interacting proteins is based on two distinct sets of data: 1) Identification of new Cell Permeable Peptide shuttles (CPP) ; 2) a new concept of PP1 predictive signature based on the simultaneous presence in most well-defined PP1 interacting proteins of these two distinct PP1c consensus docking motifs, the first well characterized canonical [RK]-x(0,1)-V-x-F and the new F-x-x-[RK]-x-[RK], that we have recently identified in some anti-apoptotic Bcl-2 proteins. Furthermore in addition to the two PP2A-derived CPPs that could be used as cargoes for macromolecule-transport, the bio-informatic identification of peptide sequences surrounding PP1c docking sites in important putative regulatory or interacting target proteins, opens the way to a new approach of phosphatase-derived drug technology. To develop this potentiality, we have established a new web site:http://pp1signature.pasteur.fr that allows the identification of putative PP1-interacting proteins containing the two distinct PP1c docking consensus motifs represented in the Swissprot library (for details see Garcia Biochimie. 85, 721-726).
PP2A represents a potential anti-tumoral target: it is established that the interaction of the adenoviral E4orf4 protein with the PP2A regulatory Ba subunit specifically induces apoptosis of transformed cells in a p53-independent manner. Based on identification of PP2A docking sites in PP2A-Ba-interacting proteins, we intend to generate penetrating biopeptides (shuttle + docking peptides) able to mimick E4orf4-signal. Putative anti tumoral peptides will be analysed ex vivo prior to be injected in mice with tumors. This approach will be realised in collaboration with the three teams MA Buendia, C.Rougeot et M.Huerre(A.Cardona), in the PTR 136.
This project should allow us: 1) to improve our understanding of the catalytic regulation of PP1 by positioning the interaction between the consensus docking motifs and PP1c. 2) to identify new functional short domains that will allow to generate new proapoptotic penetrating biopeptides mimicking the specific anti-tumoral signal mediated by PP2A- Ba-E4orf4 interaction.
Synthetic glycopeptides for anti-tumor immunotherapy
(Sylvie Bay, Christelle Ganneau and Teresa Freire)
Using a rational and specific approach of anti-tumor immunotherapy, we are developing synthetic vaccines based on carbohydrate tumor markers. We have synthesized a new immunogen : the MAG (Multiple Antigenic Glycopeptide) which carries the carbohydrate epitope (i.e. the tumor marker) associated with a T CD4+ peptide epitope. Such synthetic conjugates are particularly attractive for both their purity and accurate chemical definition, which are essential features for a safe vaccine.
We showed that the MAG system is an efficient strategy for inducing high levels of carbohydrate-specific antibodies and for increasing the survival of tumor-bearing mice, after prophylactic and therapeutic vaccinations (collaboration with R. Lo-Man et C. Leclerc, Institut Pasteur).
We have also prepared a new generation of vaccines potentially active in humans, by introducing universal CD4+ T cell epitopes. The resulting compounds raise a strong carbohydrate-specific immune response in monkeys as well as in transgenic mice for the HLA molecules.
Another part of our program concerns the synthesis of glycopeptides as immunogens for inducing specific CTL responses.
Phosphorylcholine glycoconjugates for the development of anti bacterial therapies
The program aims at developing new antibody-based therapeutic approach to fight against bacterial infections of the respiratory tract (Streptococcus pneumoniae, Neisseria meningitidis). In order to mimic the natural occurrence of the phosphoryl choline bacterial antigen, we have synthesized a glycosylated phosphorylcholine hapten. This synthon has been included in protein and biotin conjugates which will be used for generating antibodies for therapeutic and/or diagnostic applications.
Synthesis and biological activity of heterodox nucleosides
(Claudio Cadena, Laurence Dugué and Sylvie Pochet)
The main goal is to increase the repertoire of monomers that can be incorporated into DNA as well as polymers replicating in vitro and/or in vivo. Among the DNA alterations previously examined, we have focused on modifications related to the heterocyclic moiety. The impact on the recognition by DNA enzymes of these modified nucleosides (kinases, polymerases, transferases) are investigated in vitro. Several nucleobases related to oxidatively damaged nucleotides have been synthesized as well as oligonucleotides containing them. Such motifs are theoretically capable to pair with natural bases as well as with themselves. The capacity of such nucleoside analogues to act as substrates for DNA enzymes is being evaluated. These purine analogues have been used to probe the substrate recognition mechanism of the human MTH1 protein (coll. H. Kamiya, Japan).
From bacterial polysaccharides to synthetic glycoconjugate vaccines (PTR 99)
(Catherine Guerreiro, Cyrille Grandjean and Laurence Mulard)
The crucial role of bacterial polysaccharides as targets of the protective immune response elicited by the host following infection has been demonstrated on several occasions. Current studies towards the development of a polysaccharide-protein conjugate vaccine against Vibrio cholerae O1 infections involve the preparation of a panel of selected glycoconjugates based on the use of the serotype Inaba detoxified bacterial lipopolysaccharide. Evaluation of the immunogenicity of these conjugates in mice is ongoing (coll. J.-M. Fournier, Unité du Choléra et des Vibrions, Institut Pasteur).
Taking advantage of the more recent notion of "protective carbohydrate epitopes", our main goal is to develop optimal chemically defined immunogens, exposing in a multivalent fashion a combination of carbohydrate haptens (B epitopes) and appropriate T epitopes required for memory response. Shigella flexneri, a causal agent of severe dysentery, is used as a model to demonstrate the feasability of this alternative to the conventional "polysaccharide-:protein conjugate" approach for the development of multivalent vaccines.
Having characterized an immunodominant "protective" carbohydrate epitope located on the O-antigen of S. flexneri serotype 2a, glycoconjugates were designed as potential synthetic vaccines targeting homologous infections. Synthetic oligosaccharidic haptens of various lengths, representative of the above characterized epitope, were synthesized in a form suitable for coupling, and conjugated to either a T helper peptide or a protein carrier. Several of these conjugates elicited high levels of anti-LPS antibodies in mice. Evaluation of the protective efficacy of the most promising glycoconjugates is ongoing (coll. A. Phalipon, Unité de Pathogénie Microbienne Moléculaire, Institut Pasteur).
Our peptide group get specialised in the synthesis and purification of long peptides (50-70 AAs) and modified peptides.
Anti-inflammatory peptides from NEMO
NEMO (NF-kB essential Modulator) play a pivotal role in kinase activation in NF-kB signalisation pathway during immunological, inflammatory responses, oncogenesis and apoptosis. In collaboration with F. Agou and M. Véron (Unité de régulation enzymatique des activités cellulaires), we have shown that peptides from NEMO oligomerisation domain inhibit the LPS effect at micromolar range without cytotoxic effects. These peptides open the way to a new class of anti-inflammatory or anti-cancer compounds.
SDF-1a and Mip-1b/RANTES inhibit HIV viral entry in CD4+ cells via CXCR4/CCR5 co-receptors. Theses small proteins are accessible to chemical synthesis. One of the major interests in chemical synthesis of such long peptides is that we can introduce unnatural AAs, selective labelling and modification of the peptidic backbone. We designed and synthesised SDF mutants that resist to Elastase degradation, enzyme that plays an important role in the regulation of chemokine biological activity.
Keywords: synthetic vaccines, chemokines, heterodox nucleosides, in vivo selection of enzymes, directed evolution, oligonucleotides
|More informations on our web site|
|Publications 2003 of the unit on Pasteur's references database|
|Office staff||Researchers||Scientific trainees||Other personnel|
|Garnier Marie-Ange (email@example.com)||Baleux Françoise, IP (CR, firstname.lastname@example.org)
Bay Sylvie, IP (CR, email@example.com)
Garcia Alphonse, IP (CR, firstname.lastname@example.org)
Huynh-Dinh Tam, CNRS (DR1, email@example.com) : départ en retraite le 1er juillet 2003
Jestin Jean-Luc, IP (CR, firstname.lastname@example.org)
Kaminski Pierre-Alexandre, IP (CR, email@example.com)
Mulard Laurence, IP (CR, firstname.lastname@example.org)
Munier-Lehmann, INSERM (CR, email@example.com)
Pochet Sylvie, CNRS (DR2, firstname.lastname@example.org)
Vichier-Guerre Sophie, CNRS (CR2, email@example.com)
|Cadena Amaro Claudio, Thèse
Dessauge Frédéric, Thèse
Freire Gard Teresa Ines, Post-doc
Gasse Cécile, Thèse
Grandjean Cyrille, Post-doc
Guergnon Julien, Thèse
Hospital Véronique, Post-doc
|Coïc Yves-Marie, IP (Ingénieur, firstname.lastname@example.org)
Delafond Nathalie, IP (Agent de Laboratoire)
Dugué Laurence, IP (Technicienne Supérieure, email@example.com)
Dutruel Olivier, IP (Technicien Supérieur) : CDD départ mars 2003
Fantini Emmanuelle, IP (Technicienne Supérieure, firstname.lastname@example.org) : départ septembre 2003
Ganneau Christelle, IP (Technicienne Supérieure, email@example.com) : CDD
Groh François, IP (Technicien Supérieur, firstname.lastname@example.org)
Guerreiro Catherine, IP (Technicienne Supérieure, email@example.com)
Téjéro Jésus, IP (Agent de Laboratoire)
Ughetto-Monfrin Joël, CNRS (Ingénieur, firstname.lastname@example.org)