|Director : Bost Pierre-Etienne (firstname.lastname@example.org)|
The ultimate objective of the Organic Chemistry Unit is the validation of new targets and new therapeutic approaches of infectious diseases and cancer through 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. Biochemical studies of nucleoside monophosphate kinases and phosphatases correspond to these objectives. Moreover a programme of in vivo selection and in vitro directed evolution of enzymes is currently developed.
(Françoise Baleux, Yves-Marie Coïc)
With the synthesis of peptide, our group is involved in numerous collaborations within or outside the Pasteur campus.
However, an important part of our activity is dedicated to the optimisation of NEMO (IKKγ) peptides. In collaboration with F. Agou et M. Véron (unité de régulation enzymatique des activités cellulaires), we have shown that peptides from NEMO oligomerisation domain can inhibit NF-κB activation in response to pro-inflammatory (LPS, IL-1, TNF-α) or carcinogenic (PMA) stimuli (μmolar IC50 range) and that they can induce apoptosis in cancer cells in the same range. For that purpose, about 50 peptides have been synthesised during the last 3 years. These results confirm that inhibition of NF-κB pathway could lead to new classes of anti-inflammatory and anticancer drugs.
Besides long peptide synthesis (chemokines, NEMO oligomerisation domain), ß-catenin fragments (phosphorilated peptides), membrane mini-protein (PMP1) and anti-microbian peptides (Dermaseptin S4) have been synthesised for structure-function studies.
From polysaccharide antigens to anti-infectious glycoconjugate vaccines
(Laurence Mulard, Catherine Guerreiro, Ioannis Vrasidas, Cyrille Grandjean, Julien Boutet)
Capsular polysaccharides and lipopolysaccharides (LPS) are antigens of choice in the design of anti-bacterial vaccines. Although efficient polysaccharide vaccines against certain respiratory infections due to capsulated bacteria are commercially available, there is still no polysaccharide vaccine against enteric infections caused by Gram negative bacteria expressing a LPS but lacking a capsule. Research developed in the Glycochemistry team aims at overcoming this problem in the case of Vibrio cholerae O1 and Shigella flexneri, which are the major agents of cholera and bacillary dysentery, respectively. Our goal is to developed well-defined glycoconjugate vaccines based on the use of the polysaccharide moiety of the homologous LPS (pmLPS) or mimics thereof.
Another orientation in the group deals with fungal infections caused by Cryptococcus neoformans.
Vibrio cholerae O1: Relying on the description of a V. cholerae O1 serotypes Ogawa and Inaba common saccharidic protective epitope; we synthesized several V. cholerae O1 serotype Inaba pmLPS-protein conjugates. The immunogenicity of these neoglycoproteins was evaluated in mice, pointing to the high complexity of the immunological properties of the involved polysaccharide antigen (coll. J.-M. Fournier, Unité du Choléra et des Vibrions). A new method for chemical ligation was developed.
Cryptococcus neoformans: The goal is to confirm the hypothetic structure of the polysaccharide capsule that is expressed by this opportunistic fungus and to understand its immunological properties (coll. G. Janbon, Unité de Mycologie Moléculaire). Innovative efficient synthetic strategies to oligosaccharides representative of the C. neoformans capsule are being developed.
Shigella flexneri 2a: 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. Following the identification of a protective immunodominant epitope on S. flexneri 2a O-antigen, a panel of saccharidic haptens representative of this epitope and differing in length were obtained by efficient convergent multi-step syntheses. Several chemically defined glycoconjugates resulting from the site selective condensation of those synthetic haptens onto a protein carrier compatible with use in humans were synthesized. Some of these semi-synthetic glycoconjugates induce anti-LPS antibody titers in mice higher than those induced by a conventional pmLPS-protein conjugate. A pentadecasaccharide was identified as a highly potent functional mimic of the S. flexneri 2a O-antigen (coll. A. Phalipon, Unité de Pathogénie Microbienne Moléculaire). The structural bases for this mimicry are being investigated (coll. M. Delepierre, Laboratoire de RMN des Biomolécules, B. Vulliez-Le Normand, Unité d'Immunologie Structurale). A phase 1 clinical trial is being envisioned.
Phosphorylcholine glycoconjugates for the development of therapies against respiratory infections
(Christelle Ganneau, Sylvie Bay)
This program aims at developing a new antibody-based therapeutic approach against bacterial infections of the respiratory tract (Streptococcus pneumoniae, Neisseria meningitidis). In order to mimic the native bacterial target antigen, we have synthesized a glycosylated phosphorylcholine (ChoP) hapten. This synthon has been included in several synthetic or hemi-synthetic vaccine compounds. One of them, a glycoprotein, induces specific antibodies recognizing ChoP on two different bacteria (S. pneumoniae and N. meningitidis). The production of monoclonal antibodies against some of the immunogens is in progress (collaboration with P. Lafaye, Unité de Génétique et Biochimie du Développement, J-M. Alonso, Unité des Neisseria, F. Nato, PF5).
Glycoconjugates for anti-tumor immunotherapy
(Teresa Freire, Christelle Ganneau, Sylvie Bay)
In collaboration with C. Leclerc and R. Lo-Man (Unité de Recherche de Régulation Immunitaire et Vaccinologie) we are developing vaccines based on the Tn carbohydrate tumor marker. We have prepared a new synthetic immunogen, the MAG (Multiple Antigenic Glycopeptide), which is a promising vaccine candidate against breast, lung or prostate cancer.
To further extend the scope of our approach to clinical applications, we have developed new enzymatic syntheses of glycoconjugates. The resulting vaccines are based on the tumor-associated mucin MUC6 and display high densities of the Tn antigen. Their immunological properties are currently under investigation.
Nucleoside monophosphate kinases - synthesis and antimycobacterial activity of nucleoside analogs
(Hélène Munier-Lehmann, Khaled Benkali, Cécile Gasse, Laurence Dugué, Valérie Huteau, Sylvie Pochet)
The nucleoside monophosphate kinases (NMPKs) catalysed the phosphorylation of nucleoside monophosphate into their corresponding diphosphate derivatives and are characterized by a high specificity for the nucleoside monophosphate substrate. The five NMPKs from M. tuberculosis have been overexpressed, purified and characterized. These enzymes exhibit distinct characteristic from their eukaryotic homologs. The corresponding genes are essential, thus these enzymes were selected as potential targets for antituberculosis therapy. As the 3D-structure of TMPK was the first to be solved, complementary approaches have been selected for the search of inhibitors: synthesis of natural substrate analogues, in silico screening of chemical libraries, design of non nucleosidic compounds. A first series of specific inhibitors were found to be active on in vitro M. tuberculosis culture. Similar approaches would be apply to the other NMPKs.
Nucleoside analogs as potent inhibitors of viral replication
(Laurence Dugué, C. Cadena, Sylvie Pochet)
Accumulation of mutations by the action of mutagenic agents may lead to viral extinction, in a transition termed virus entry into error catastrophe. Recent experiments with nucleoside analogs validate this new approch to treating infection of RNA viruses. We have synthesized following an original parallel synthetic scheme, a large series of nucleosides and deoxynucleosides having as nucleobase different heterocycles capable to pair with more than one of the canonical bases. The capacity of these nucleoside analogs to promote viral extinction would be evaluated in collaboration with E. Domingo(Madrid) and M.-A. Martinez (Badalona).
Substrate specificity of vaccinia virus thymidylate kinase
(Laurence Dugué, Cécile Gasse, Sylvie Pochet)
In collaboration with D. Deville-Bonne (Institut J. Monod, Université Pierre et Marie Curie-Paris 6), thymidylate fluorescent analogs have been synthesized as fluorescent probes of the dTMP binding site of thymidylate kinase from Vaccinia virus. A first series of nucleotide analogues have been synthesized and used to characterize the substrate specificity of the enzyme. Specific inhibitors and alternate substrates of vvTMPK should contribute to virus replication inhibition.
Isoniazid is currently used for the treatment of tuberculosis across the world. Its mechanism of action is still the subject of research. It is only in the last 7 years that it was demonstrated that this drug acts via a metabolic activation by a mycobacterial catalase to give an isoniazid-NADH adduct. Remarkably, this adduct (or a related structure) is the actual inhibitor of the mycobacterial enoyl reductase which thus leads to the bacterium growth arrest. We are currently trying to synthesize and test compounds that would resemble this isoniazid-NADH adduct and remain active on the Mycobacterium tuberculosis enoyl reductase.
The PP1/PP2A protein phosphatases are new potential therapeutic targets
(Alphonse Garcia, Julien Guergnon,Virginie Maire, Angélique Godet)
We recently proposed a new concept for a phosphatase-derived drug technology (DPT) which is based on the design of penetrating peptides able to interact with PP1 or PP2A holoenzymes and able to specifically perturbate PP1/PP2A-directed intracellular pathways (Guergnon et al. Mol.Pharmacol in press). Based on this concept we are currently analyzing the role PP2A in apoptotic pathways mediated by penetrating peptides originated from the two viral, HIV-1-Vpr and canine adenovirus E4orf4, proteins.
Directed enzyme evolution using selection
(Jean-Luc Jestin, Pierre-Alexandre Kaminski, Sophie Vichier-Guerre)
Whereas classical directed enzyme evolution strategies make use of screening, we develop selections to analyse simultaneously the catalytic activity of more than 10e8 distinct proteins. The selections for catalysis are set up in vivo using engineered Escherichia coli strains and in vitro using a chemistry of filamentous phages.
Thermostable DNA polymerases endowed with two catalytic activities, reverse transcriptase activity and DNA dependent DNA polymerisation have been obtained. Further, application of this enzyme engineering strategy for the isolation of glycosyl transferases with new substrate specificities is pursued in order to facilitate the synthesis of glycoconjugate vaccines. Finally, alterations of the specificity of N-deoxyribosyltransferases acting on nucleosides have been obtained by in vivo selection.
Beside the applications of these engineered enzymes, a special focus is put on relations between the sequences, structures and catalytic activities of these enzymes.
Keywords: glycopeptide, glycoprotein, cancer, immunotherapy, vaccines, carbohydrate, enteric infections, peptide chemistry, chemokines, heterodox nucleosides, antimycobacterial agents, in vivo selection of enzymes, phage display, directed evolution, oligonucleotides, nucleoside monophosphate kinases, PP1/PP2A, apoptosis
|Publications 2005 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 (CL, firstname.lastname@example.org)
Janin, Yves, CNRS (CR1, email@example.com)
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, Hélène, INSERM (CR1, email@example.com)
Pochet, Sylvie, CNRS (DR2, firstname.lastname@example.org)
Vichier-Guerre, Sophie, CNRS (CR1, email@example.com)
|Auberger Nicolas, Master 1 student
Benkali Khaled, Master 2 student
Boutet Julien, PhD student
Caron Amélie, Master 1 student
Castex Anaïs, Master 1 student
Dittoo Aurélia, Master 1 student
Fouillet Delphine, Master 1 student
Freire Gard Teresa Ines, Postdoc
Gasse Cécile, PhD student
Grandjean Cyrille, Postdoc
Guergnon Julien, PhD student
Konto Ghiorghi Yoan, Master 2 student
Mahiddine Karim, Master 2 student
Naah Marielle, Master 1 student
Nesme Olivier, Master 2 student
Ouarti Naïma, Master 2 student
Prado Soizic, Postdoc
Vekhoff Pierre, Master 2 student
Vrasidas Ioannis, Postdoc
|Bonhomme Frédéric, CNRS (Engineer, firstname.lastname@example.org)
Coïc Yves-Marie, IP (Engineer, email@example.com)
Delafond Nathalie, IP (Laboratory worker, firstname.lastname@example.org)
Dugué Laurence, IP (Technician, email@example.com)
Ganneau Christelle, IP (Technician, firstname.lastname@example.org)
Groh François, IP (Technician, email@example.com)
Guerreiro Catherine, IP (Technician, firstname.lastname@example.org)
Huteau Valérie, IP (Technician, email@example.com)
Maire Virginie, IP (Engineer, firstname.lastname@example.org) : CDD
Téjéro Jésus, IP (Laboratory worker, email@example.com)