|Organic Chemistry - CNRS URA2128|
|HEAD||Dr POCHET Sylvie / firstname.lastname@example.org|
|MEMBERS||BONHOMME Frédéric (IE2, CNRS) DELAFOND Nathalie (Agt Labo) DUFOUR Emilie (stagiaire) DUGUE Laurence (Techn Sup, IP) Dr DUPOUY Christelle (post-doc CNRS) GARNIER Marie-Ange (secrétariat) FERRANDEZ Anne-Claire (stagiaire) HUTEAU Valérie (Techn Sup, IP) Dr KAMINSKI Alexandre (CR, IP)
Dr LUPAN Alexandru (post-doc, IP) LUO Wen (stagiaire) Dr MORELLATTO Laurence (post-doc CNRS) Dr MUNIER-LEHMANN Hélène (CR1, INSERM) Dr POCHET Sylvie (DR2, CNRS) TEJERO Jesus (Agt Labo) Dr VICHIER-GUERRE Sophie (CR1, CNRS) ZHANG Che (stagiaire)
The research projects of the Organic Chemistry Unit lies at the interface of Chemistry Biology and are mainly focused on the design and synthesis of molecules that interfere with nucleoside metabolism enzymes, and the characterization of unexplored targets related to this metabolism. The research aims to a better understanding of biochemical and biological mechanisms and to the development of molecules for detecting and treating diseases.
We combine tools and methods derived from chemistry (e.g. chemical and enzymatic synthesis of nucleoside analogs) as well as from biochemistry and molecular biology (e.g. protein expression and purification, biochemical characterization, in vivoenzyme selection). Since 2007, a versatile screening plateform was set-up allowing automated enzymatic and cellular tests on chemical libraries. Current research projects center around the following areas:
Synthesis of nucleoside analogs through enzymatic process
The enzymatic synthesis of modified nucleosides through a transglycosylation reaction catalyzed by nucleoside phosphorylases or deoxyribosyltransferases provides several advantages over chemical routes, such as regio and stereoselectivity and environmentally clean one pot reactions. These enzymes are successfully applied to the preparation of nucleoside analogs. In order to enlarge the number of substrates differing either by their heterocycle or by their sugar counterpart, we have cloned and expressed NDT from different Lactobacilli. Specific applications such as transfer of 2’,3’-dideoxyribose and or 2’,3’-didehydroribose between bases, required tailored enzymes that were obtained by in vivo selection in appropriate genetically engineered strains.
With the emergence of antibiotic resistance worldwide, the search for new antibacterial compounds has become increasingly important. With the aim to identify new inhibitors based on novel mechanisms of action, we selected key bacterial enzymes as novel potential targets, and applied different strategies (structure-based drug design, in silicoscreening, functional screening of academic and commercial libraries, fragment-based approaches) to define inhibitors.
Nucleoside monophosphate kinases from M. tuberculosis
Nucleoside monophosphate kinases (NMPKs) are ubiquitous enzymes, which catalyze the reversible transfer of phosphoryl group from ATP to an NMP, and are essential for cell division, growth and metabolism. The five NMPKs of M. tuberculosisand their human counterparts have been biochemically characterized, and their 3D-structures solved. The last years were mainly devoted to the search of potent and selective inhibitors of TMPK of M. tuberculosis. Based upon the 3D structure of M. tuberculosisTMPK, we have designed molecules as substrate mimics to generate competitive inhibitors, and thus identified original molecules acting in vitroas selective inhibitors of TMPKmt and mycobacterial strains.
Nicotinamide adenosine kinase from Gram-positive bacteria
NADKs are essential for growth in many bacteria including various human pathogens. They catalyze the phosphorylation of NAD to produce NADP, a cofactor involved in energy metabolism and biosynthetic pathway. We have determined the crystal structures of NADK from the human pathogen L. monocytogenesin complex with two natural ligands, NAD and NADP, as well as with various adenosine derivatives. In particular, we described the first complex of a NADK with an non-natural inhibitor DTA, the first synthetic competitive inhibitor of a NADK described so far. These results constitute the starting point for the design of new inbibitors following the fragment-based drug appproach.
Characterisation of a new enzymatic activity
The human gene rcl(c6orf108) is regulated by c-Myc and overexpressed in several cancers. Rcl is assumed to be involved in cell growth and/or cell proliferation. The recent correlation of the Rcl expression and the increasing tumor grade reinforced this hypothesis. Our characterization of Rcl (member of the N-deoxryribosyltransferase family) revealed a novel enzyme activity, namely a 2’-deoxynucleoside 5’-monophosphate N-glycosidase. The structure-activity relationship of this new, recently patented, family of nucleoside catabolic enzymes of potent therapeutic interest is under study.
Our versatile automated platform (TECAN Freedom EVO® platform) with open-architecture software capable of integrating off-the-shelf peripherals is now operational and available for collaborative projects. Diverse chemical libraries including the French National Chemical Library (“Chimiothèque Nationale”), commercial and in-house libraries are evaluated for their potential activity on purified enzymes from our laboratory and on cellular assays (in collaboration with partners from Institut Pasteur).
Keywords: Antibacterial Agents, Medicinal Chemistry, Nucleosides, Nucleoside metabolism enzymes, Chemical library screening. Enzyme in vivo selection. Chemo-enzymatic approaches
1. Kaminski, P. A.; Dacher, P.; Dugué, L.; Pochet, S. (2008) In vivo reshaping the catalytic site of nucleoside 2 '-deoxyribosyltransferase for dideoxy- and didehydronucleosides via a single amino acid substitution. Journal of Biological Chemistry,283, 20053-20059.
2. Gasse, C.; Douguet, D.; Huteau, V.; Marchal, G.; Munier-Lehmann, H.; Pochet, S. (2008) Substituted benzyl-pyrimidines targeting thymidine monophosphate kinase of Mycobacterium tuberculosis: Synthesis and in vitro anti-mycobacterial activity. Bioorganic & Medicinal Chemistry,16, 6075-6085.
3. Poncet-Montange, G.; Assairi, L.; Arold, S.; Pochet, S.; Labesse, G. (2007) NAD Kinases Use Substrate-assisted Catalysis for Specific Recognition of NAD. Journal of Biological Chemistry, 282, 33925-33934.
4. Van Daele, I., Munier-Lehmann, H., Froeyen, M., Balzarini, J. & Calenbergh, S. V. (2007) Rational design of 5'-thiourea-substituted alpha-thymidine analogues as thymidine monophosphate kinase inhibitors capable of inhibiting mycobacterial growth. Journal of Medicinal Chemistry, 50, 5281-5292.
5. Y. K. Ghiorghi, K. I. Zeller, C. V. Dang, P. A. Kaminski (2007). The c-Myc target gene Rcl (C6orf108) encodes a novel enzyme, deoxynucleoside 5'-monophosphate N-glycosidase. Journal of Biological Chemistry, 282, 8150-8156.
Activity Reports 2009 - Institut Pasteur
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