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  Director : Huynh-Dinh Tam (hdt@pasteur.fr)


  abstract

 

The Unit of Organic Chemistry is involved in three important classes of biological compounds : nucleosides or nucleotides, peptides and sugars. The synthesis of these molecules permit the study of their structures and interactions ; the final goal is the development of synthetic vaccines or enzyme inhibitors, in collaboration with other units on the campus. Two groups in the Unit are involved in a program aiming to the in vivo selection or in vitro directed evolution of enzymes.



  report

cale

Chemokines/HIV

Chemokines SDF-1a and Mip-1b/RANTES inhibit HIV entry in CD4+ cells by CXCR4and CCR5 co-receptors. These small proteins (70 AAs) are readily available by chemical synthesis, which allows selective labelling by fluorochromes or biotin, generating still biological actives molecules. We demonstrated that Leukocyte Elalastase negatively regulates SDF/CXCR4 binding and functions by amino-terminal processing of SDF and CXCR4. Chemical modification of the Elastase-targetted peptide bond generates a chemokine surviving Elastase degradation, maintaining its inhibitory capacity.

Structure, folding of peptide and membrane interaction

*NMR analysis shows that most of the structural elements of the globular domain of murine prion protein form fibrils with predominant -sheet structure.

*PMP1 is a small single-spanning membrane protein functioning as a regulatory subunit of the yeast plasma membrane H+-ATP-ase. Despite its very hydrophobic character, its chemical synthesis was performed. NMR analysis in DPC/POPS micelles reveals that the clustering of POPS molecules is promoted from a stabilised framework obtained by coupling the PMP1 helix groove to a POPS sn-2 acyl lipid chain.

Glycosylated epitopes secreted by Mycobacterium tuberculosis (Françoise Baleux/Laurence Mulard)

Recent data point out the importance of secreted mannosylated proteins, among which Apa, as immunodominant antigens upon infection by M. tuberculosis. Glycopeptide dominant epitopes are anticipated. However, although, the amino acid sequence of potential targets are known, the biochemical approach failed to reveal their precise glycoforms. Thus, taking advantage of the flexibility of both carbohydrate chemistry and peptide solid phase synthesis, selected mannosylated peptides were synthesized. One of the synthetic glycopeptides was identified as an immunodominant epitope for the Apa protein.

From bacterial polysaccharides to synthetic glycoconjugate vaccines (Laurence Mulard/Françoise Baleux)

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. The more recent notion of "protective carbohydrate epitopes" opened new opportunities in the field of anti-bacterial glycoconjugate vaccines. Indeed, derived from the concept of hapten, our goal is to develop optimal chemically defined immunogens, exposing in a multivalent fashion a combination of such "protective carbohydrate epitopes" (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.

The optimisation of synthetic glycoconjugates as potential Shigella vaccines relies on an increased understanding of the specificity of the host's immune response elicited upon infection. For that purpose, complex oligosaccharides representative of frame-shifted fragments of the surface polysaccharide of S. flexneri serotypes 2a and 5a, were synthesized and their binding to protective monoclonal antibodies was studied. Based on the knowledge gained from this rational approach, glycoconjugates were designed as potential synthetic vaccines targetting S. flexneri infections. Indeed, fragments specific for S. flexneri 2a O-antigen were synthesized in a form suitable for coupling, and selected short haptens were conjugated to a T helper peptide. Immunogenicity studies are in progress.

Synthesis of nucleotide analogs as potential antivirals (Laurence Mulard)

The design of new potent antivirals, based on the study of two key steps of the viral replication inhibition, is our main objective. Recent data demonstrated that alphaborano triphosphate derivatives of known antivirals were better inhibitors of the HIV reverse transcriptase than their triphosphate counterparts. The next step is to find an -borano nucleotide pro-drug that could favor the entry of the borano derivative into cells and that would bypass the rate-limiting steps of intracellular phosphorylation. Towards this aim, several potential pro-drugs of d4T borano monophosphate were synthesized. The synthesis of a set of analogs in the AZT series allowed stability studies of the -boranophosphate moiety to be performed both in cell culture media and at various pH.

Synthetic glycopeptides for anti-tumor immunotherapy (Sylvie Bay)

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 (Sylvie Bay)

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 (Alexandre Kaminski / Sylvie Pochet).

The goal of our research group is to increase the repertoire of monomers that can be incorporated into DNA as well as polymers replicatable in vitro and/or in vivo. Two kinds of DNA alterations are currently investigated : the first modification concerns the heterocyclic moiety. A combinatorial approach aiming to produce of a great number of nucleosides triphosphates in one step was developped in order to find new monomers having highly specific or ambiguous base-pairing schemes, or acting as inhibitors of replication. The second modification concerns the sugar conterpart: HNAs are conformationally constrained furanose mimicking nucleic acids in which the sugar part is replaced by 1,5-anhydrohexitol. The capacity of such polymers to support genetic information transfer was investigated in vitro and in vivo.

In order to enlarge the repertoire of unnatural nucleosides available by enzymatic process, genes coding for N-deoxyribosyltransferase from different lactobacilli were isolated. The catalytic properties of the encoded enzymes are currently under investigation. Apart from the natural enzymes, catalysts with altered substrate specificity are evolved by combining in vitro mutagenesis and in vivo selection in auxotrophic strains of E. coli. As a first demonstration, a dideoxyribosyltransferase was obtained by selecting N-deoxyribosyltransferase mutants for their capacitiy to restore the uracil auxotrophy of an E. coli strain with dideoxyruridine as a source of uracil.

A project aiming to obtain potent inhibitors of M. tuberculosis growth was initiated. Nucleoside monophosphate kinases (TMPK, UMPK) which are essential for the bacterial growth were chosen as therapeutic targets. Futhermore, biochemical characterization of TMPK revealed new structural and catalytic features, which renders this protein a good target for antituberculosis drugs. The selected approach will be the structure-based design followed by the synthesis and the determination of the inhibitory effect of the designed compounds both in vitro and in vivo.

Directed enzyme evolution (Sophie Vichier-Guerre/Jean-Luc Jestin)

In our group, we develop a chemistry of Inovirus to isolate catalysts from repertoires of more than 108 proteins. The directed enzyme evolution strategy includes in vitro selections of proteins for catalysis and in vivo amplification of the corresponding genes.

Remarkable results include : (i) the isolation of a catalyst among more than 108 other proteins (ii) the generalisation of the in vitro selection for catalytic activity to substrate cleaving reaction in addition to the synthetic reactions. (iii) the generalisation of the in vitro selection for catalytic activity to other protein display systems (iv) enrichment factors above 104 per cycle, the highest described so far for in vitro selections according to catalytic activity, whatever the display system.

This directed enzyme evolution strategy has applications in enzyme engineering and in therapeutic chemistry as well as in functional genomics for the identification of therapeutic targets.

Oligonucleotides (Tam Huynh-Dinh)

In a collaborative work in the field of DNA micro arrays, we have developped two methods of covalent coupling at the 3' or the 5' end of oligonucleotides on glass slides with a hydrazone bond. This work aims to the easy production of low-cost DNA arrays with a high hybridization signal.

The synthesis of DNA or RNA fragments was carried out for structural studies (DNA with Hoogsteen base-pairing, RNA loops, triple helices) and nucleic acids-proteins interactions (HIV dimerization complex-nucleocapside). Synthesis of iRNA was also done for the control of gene expression.

Keywords: synthetic vaccines, chemokines, heterodox nucleosides, in vivo selection of enzymes, directed evolution, oligonucleotides



  publications

puce Publications of the unit on Pasteur's references database


  personnel

  Office staff Researchers Scientific trainees Other personnel
  Garnier Marie-ange (mag@pasteur.fr) Baleux Françoise, IP (baleux@pasteur.fr)

Bay Sylvie, IP (sbay@pasteur.fr)

Huynh-Dinh Tam, CNRS (hdt@pasteur.fr)

Jestin Jean-Luc, IP (jjestin@pasteur.fr)

Kaminski Pierre-Alexandre, IP (akaminsk@pasteur.fr)

Mulard Laurence, IP (lmulard@pasteur.fr)

Pochet Sylvie, CNRS (spochet@pasteur.fr)

Vichier-Guerre Sophie, CNRS (svichier@pasteur.fr)

Bélot Frédéric, Post-doc

Bousserouel Hadjira, DEA

Cadena Amaro Claudio, Thèse

Cocoletzi Avila Brenda, DEA

Perrier Sandrine, Thèse

Strobel Heike, Post-doc

Coïc Yves-Marie, IP (ymcoic@pasteur.fr)

Delafond Nathalie, IP

Dugué Laurence, IP (ldugue@pasteur.fr)

Dutruel Olivier, IP (odutruel@pasteur.fr)

Fantini Emmanuelle, IP (efantini@pasteur.fr)

Gouyette Catherine, IP (oligos@pasteur.fr)

Groh François, IP (fgroh@pasteur.fr)

Guerreiro Catherine, IP (cguerrei@pasteur.fr)

Helynck Olivier, IP

Huteau Valérie, IP (vhuteau@pasteur.fr)

Raghouber Josiane, IP

Ughetto-Monfrin Joël, CNRS (ughetto@pasteur.fr)

Vieira Da Silva Francisco, IP


Activity Reports 2002 - Institut Pasteur
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