|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 the physical properties and activities of these compounds in various biological systems. The ultimate objective includes the validation of new targets and new therapeutic approaches of infectious diseases and cancer. Biochemical studies of nucleoside monophosphate kinases and phosphatases correspond to these objectives. Moreover, a programme of in vivo and in vitro selections for directed evolution of enzymes is currently developed.
1. Peptide synthesis
The peptide group has specialised in the synthesis and purification of long peptides (50-70 AAs) and modified peptides.
(Françoise Baleux, Yves-Marie Coïc)
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 rely on the introduction of unnatural AAs and selective labelling and modification of the peptidic backbone. We designed and synthesised SDF mutants that resist to elastase degradation, an enzyme playing an important role in the regulation of chemokine biological activity. In order to improve the stability of SDF-1a vs. elastase, analogues containing the modified peptide bonds (CH2-NH, CH2-CO) or D-amino-acids have been synthesised at leu5. More recently we have demonstrated that oligosaccharides like heparin or heparin sulphate protect SDF-1a from proteolysis.
Anti-inflammatory peptides from NEMO
(Françoise Baleux, Yves-Marie Coïc)
NEMO (NF-kB essential Modulator) plays 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. About 30 peptides of various lengths (30 to 60 AAs) were prepared. These peptides may open the way to a new class of anti-inflammatory or anti-cancer compounds. More recently a 84 AA peptide, fully water soluble and interfering with the whole domain of CC2-LZ, 2 coil-coil sub-domain of NEMO, has been synthesised for further NMR and X-Ray studies.
2. From bacterial carbohydrates to glycoconjugate vaccines
Polysaccharide-protein conjugates as potential vaccines against cholera
(Laurence Mulard, Cyrille Grandjean)
In collaboration with J-M. Fournier's team (Unité du Choléra et des Vibrions) our goal is to develop a polysaccharide-protein conjugate vaccine against Vibrio cholerae serogroup O1. Available knowledge indicates that the polysaccharide moiety of V. cholerae O1 lipopolysaccharide (LPS) is the major target of the host's protective immune response upon infection. However, LPS toxicity in humans prevents its direct use in vaccines. Thus, current studies involve the preparation of a panel of selected glycoconjugates based on the use of the V. cholerae O1 serotype Inaba detoxified LPS. All the synthesized polysaccharide-protein conjugates were found antigenic. In combination with structural analysis, improving their immunogenicity is our next goal.
Chemically defined oligosaccharide conjugates as potential vaccines against bacillary dysentery
(Laurence Mulard, Catherine Guerreiro, Cyrille Grandjean)
Taking advantage of the 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.
Here, Shigella flexneri is used as a model to demonstrate the feasibility of using synthetic oligosaccharide conjugates for the development of multivalent vaccines. Indeed, since protection is known to be serotype-specific, efficient prevention of severe dysentery caused by S. flexneri, requires at least a trivalent vaccine.
Having characterized an immunodominant "protective" carbohydrate epitope located on the O-antigen portion of S. flexneri serotype 2a LPS, oligosaccharide haptens of various lengths, representative of the above characterized epitope, were synthesized in a form suitable for site-specific coupling, and conjugated to either a universal T helper peptide (fully synthetic conjugates, coll. F. Baleux) or a protein carrier (semi-synthetic conjugates) compatible with human use. In collaboration with A. Phalipon (Unité de Pathogénie Microbienne Moléculaire) we showed that several semi-synthetic glycoconjugates elicited high levels of both anti-oligosaccharide and anti-LPS antibodies in mice. In addition, the anti-LPS antibody response was shown to be protective against homologous infection in mice. Interestingly, the protective efficacy was highly dependent on the length of the carbohydrate hapten. Besides, our immunogenicity/protection data suggest that a synthetic pentadecasaccharide acts as a powerful mimic of the natural O-antigen. In order to confirm this hypothesis, conformational analysis of the S. flexneri 2a O-antigen and synthetic fragments thereof is ongoing (coll. M. Delepierre, Laboratoire de Résonance Magnétique Nucléaire des Biomolécules). A clinical trial is envisioned.
Phosphorylcholine glycoconjugates for the development of therapies against respiratory infections
(Sylvie Bay, Christelle Ganneau)
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 (GalNac-ChoP). This synthon has been included in protein and biotin conjugates. The glycoprotein induces specific antibodies recognizing ChoP on two different bacteria (Streptococcus pneumoniae and Neisseria meningitidis) (collaboration with P. Lafaye, Unité de Génétique et Biochimie du Développement, and J-M. Alonso, Unité des Neisseria).
3. Synthetic glycopeptides for anti-tumor immunotherapy
(Sylvie Bay, Teresa Freire, Christelle Ganneau)
In collaboration with C. Leclerc and R. Lo-Man (Unité de Biologie des Régulations Immunitaires) 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.
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 transgenic mice for the HLA molecules as well as in primates. Primate antibodies induce an antibody-dependent cytotoxicity on human cell lines.
To further extend the scope of our approach to clinical applications, new enzymatic synthesis of glycoconjugates are currently developed.
4. Synthesis and properties of unnatural nucleosides
(Claudio Cadena, Laurence Dugué et Sylvie Pochet)
As a part of our research program on nucleoside analogues to provide structures with altered properties, we have focussed on the design and synthesis of modified nucleosides that can be incorporated into DNA as well as polymers replicating in vitro. 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) 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.
5. Nucleoside monophosphate kinases; synthesis of inhibitors and antimycobacterial activities of nucleoside analogues
(Hélène Munier-Lehmann, Edward Seclaman, Sylvie Pochet, Laurence Dugué, Cécile Gasse)
The nucleoside monophosphate kinases (NMPKs) from M. tuberculosis were selected as potential targets for antituberculosis therapy. As the 3D-structure of TMPK has been resolved, 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.
Those having micromolar inhibitory activity have been evaluated on mycobacteria cultures. Among benzyl-substituted thymine derivatives two compounds have shown inhibition of the growth of BCG and H37Ra strains.
6. Apoptosis and new therapetic approaches based on PP1/PP2A protein phosphatases
(Alphonse Garcia, Julien Guergnon, Véronique Hospital, Frédéric Dessauge, 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/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.
The Phosphatase group intend to deliver ex vivo and in vivo peptides with sequence surrounding PP1/PP2A binding sites identified in important regulatory interacting proteins, based on two distinct sets of data: i) identification of new cell permeable peptide shuttles (CPP) ii) 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.
PP2A represents a potential anti-cancer target: 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 to improve our understanding of the catalytic regulation of PP1 by positioning the interaction between the consensus docking motifs and PP1c and to identify new functional short domains in order to generate new proapoptotic penetrating biopeptides mimicking the specific signal mediated by PP2A- Ba/E4orf4 interaction.
7. Directed enzyme evolution using selection
(Jean-Luc Jestin, Pierre-Alexandre Kaminski, Sophie Vichier-Guerre, Stéphane Ferris)
Whereas 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 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 novel catalytic activities such as reverse transcriptase activities 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: glycoconjugates, vaccines, peptide chemistry, chemokines, heterodox nucleosides, antimycobacterial agents, in vivo selection of enzymes, directed evolution, oligonucleotides, nucleoside monophosphate kinases, phosphatases
|More informations on our web site|
|Publications 2004 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 (CR2, email@example.com)
Cadena Amaro Claudio, Thèse
Dessauge Frédéric, Thèse
Freire Gard Teresa Ines, Post-doc
Gasse Cécile, Thèse
Gourdier Isabelle, Post-doc
Grandjean Cyrille, Post-doc
Guergnon Julien, Thèse
Morel Mathieu, Stage d'Ingénieur
Prado Soizic, Post-doc
Seclaman Edward, Post-doc
Vekhoff Pierre, Master2
|Coïc Yves-Marie, IP (Ingénieur, firstname.lastname@example.org)
Delafond Nathalie, IP (Agent de Laboratoire)
Dugué Laurence, IP (Technicienne Supérieure, email@example.com)
Ferris Stéphane, IP (Technicien Supérieur, firstname.lastname@example.org)
Ganneau Christelle, IP (Technicienne Supérieure, email@example.com)
Groh François, IP (Technicien Supérieur, firstname.lastname@example.org)
Guerreiro Catherine, IP (Technicienne Supérieure, email@example.com)
Maire Virginie, IP (Ingénieur, firstname.lastname@example.org) : CDD
Téjéro Jésus, IP (Agent de Laboratoire) : CDD
Ughetto-Monfrin Joël, CNRS (Ingénieur, email@example.com)