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  Chimorg


  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

From bacterial polysaccharides to synthetic glycoconjugate vaccines (L. 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. 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. A strategy to glycoconjugates as potential synthetic vaccines against S. flexneri was designed based on the knowledge gained from this rational approach. Thus, selected short fragments specific for S. flexneri 2a O-antigen were synthesized in a form suitable for further conjugation to a carrier.

Glycosylated epitopes secreted by Mycobacterium tuberculosis (L. Mulard, F. Baleux).

Recent data point out the importance of secreted mannosylated proteins 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. Their evaluation as potential M. tuberculosis epitopes is ongoing.

Synthesis of nucleoside and nucleotide analogs as potential antivirals (L. 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 viral replication than their triphosphate counterparts. The next step is to find an a-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 understanding of the contribution of the functional groups of a known antiviral to its activity may help the design of analogs of improved potency and decreased toxicity. For this purpose, several analogs of Ribavirin triphosphate were synthesized. Modifications pointed out the crucial role of the 3'-hydroxyl group of native Ribavirin.

Peptide synthesis (F. Baleux)

Chemokines/ HIV

Chemokines SDF-1 a and Mip-1 b/RANTES inhibit HIV entry in cells via CD4 and CXCR4/CCR5 co-receptors. These 70 AAs proteins are accessible to chemical synthesis that moreover allows selective tagging (fluorochromes, biotin) of AAs not implicated in chemokines biological activity, thus leading to still active proteins.

In addition to the key role of SDF/HS interaction, we point out the proteolytic regulation of SDF activity by two serine proteases. The N-Terminal truncated chemokines generated lost their biological activity. Chemical modification of the serine proteases targeted peptide bond allowed us to generate chemokines resistant to these serine proteases.

Peptide structure, folding and lipids interaction

PMP1 is a small membrane protein functioning as a regulatory subunit of the yeast H+-ATPase. Despite its high hydrophobic character, PMP1 chemical synthesis was achieved. PMP1 was chosen as a convenient candidate for exploring the molecular mechanisms underlying the overall lipid-peptide interaction network.

PrP a-helixes intrinsic structural propension analyses in correlation with amyloid aggregation potency are under investigation by introduction of mutations within these a-helices.

Synthesis and biological activity of heterodox nucleosides (A. Kaminski, S. 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 triphosphate in one step was developped in order to find monomers having highly specific or ambiguous base-pairing schemes, or new inhibitors to replication. The second modification concerns the sugar counterpart. 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 selected proteins from M. Tuberculosis started. Nucleoside monophosphate kinases (TMPK, UMPK and STPKs) which are essential for the bacterial growth were chosen as therapeutic targets. 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 on the recombinant targets and in vivo on M. tuberculosis. Biochemical characterization of MT TMPK revealed new structural and catalytic features, which renders this protein a good target for antituberculosis drugs.

Oligonucleotides (T. 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 oligonucléotides on glass slides. A study of the silanization of the glass surfaces as been also done. All 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 of the loops and triple helices and nucleic acids-proteins interactions (HIV dimerization complex-nucleocapside). Synthesis of iRNA was also done for the control of gene expression.

Synthetic glycopeptides for anti-tumor immunotherapy (S. 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 are currently elaborating new generations of MAG vaccines more powerful and potentially active in humans, by introducing CD8+ T cell epitopes as well as universal CD4+ T cell epitopes. The immunogenicity of first linear model compounds are under investigation in transgenic mice for the HLA molecules. This approach will be next extended to the MAG system using a fragment ligation strategy.

Another part of our program concerns the synthesis of glycopeptides i) as immunogens for inducing specific CTL responses, ii) as substrates for studying various glycosyl transferases specificity, iii) for Tn-binding proteins specificity studies (lectins and antibodies).

Directed enzyme evolution (J.L. Jestin)

To direct evolution of enzymes, we develop a Chemistry of Inovirus to select proteins for catalytic activity. We aim to establish a strategy for the isolation of enzymes for given chemical reactions by in vitro selection.

Our in vitro selection of proteins for catalytic activity is an affinity selection for the reaction product crosslinked to the phage-enzyme that catalysed the reaction from substrate to product. Enzymes are expressed on the surface of filamentous phage to link genotype and phenotype.

DNA-dependent DNA-polymerases have been expressed on the surface of bacteriophage. Using the phagemid system, only one phage in thousand displays a polymerase. A signal sequence library was designed and submitted to in vitro selection for the optimisation of polymerase display on phage. Expression levels of 1 in 5 have been achieved, suggesting that isolation of an appropriate signal sequence might be a general means of improving the display on filamentous phage.

Currently, we aim to generalise the in vitro selection for catalytic activity to other enzymes and to isolate catalysts for given chemical reactions from synthetic or natural protein libraries.



  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 (CR, baleux@pasteur.fr)

Bay Sylvie, IP (Assistante, sbay@pasteur.fr)

Huynh-Dinh Tam, CNRS (DR1, hdt@pasteur.fr)

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

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

Mulard Laurence, IP (CR, lmulard@pasteur.fr)

Pochet Sylvie, CNRS (CR1, spochet@pasteur.fr)

Vichier-Guerre Sophie, CNRS (CR2, 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 (Ingénieur, ymcoic@pasteur.fr)

Delafond Nathalie, IP (Agent de Laboratoire)

Dugué Laurence, IP (Technicienne Supérieure, ldugue@pasteur.fr)

Dutruel Olivier, IP (Technicien supérieur, odutruel@pasteur.fr)

Fantini Emmanuelle, IP (Technicienne Supérieure, efantini@pasteur.fr)

Gouyette Catherine, IP (Ingénieur, oligos@pasteur.fr)

Groh François, IP (Technicien supérieur, fgroh@pasteur.fr)

Guerreiro Catherine, IP (Technicienne Supérieure, cguerrei@pasteur.fr)

Helynck Olivier, IP (Technicien supérieur)

Huteau Valérie, IP (Technicienne Supérieure, vhuteau@pasteur.fr)

Raghouber Josiane, IP (Agent de Laboratoire)

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

Vieira Da Silva Francisco, IP (Agent de Laboratoire)


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