Our research focuses mainly on designing and synthesizing new chemical entities able to interfere with nucleoside metabolism enzymes, and characterizing unexplored proteinsrelated to this metabolism. Our studies aim to provide a more in-depth understanding of biochemical and biological mechanisms, and to discover compounds for treating diseases. Our complementary skills allow us to combine tools and methods derived from organic chemistry (e.g. chemical and enzymatic synthesis of nucleoside analogs) and from biochemistry and molecular biology (e.g. protein expression and purification, biochemical characterization, in vivoenzyme selection). The unit manages the screening activity devoted to investigating the effect of small molecules mainly in the field of anti-infectious and anti-cancer agents.

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. In order to enlarge the number of substrates differing either by their heterocyclic or by their sugar counterpart, tailored NDT variants from different Lactobacilli were obtained by in vivoselection in appropriate genetically engineered strains. These enzymes are successfully applied to the preparation of a new family of nucleoside analogs.

Antibacterial agents

The emergence of multidrug-resistant pathogens has led to the need for new antibiotics acting on previously unexplored targets. We have selected and characterized key bacterial enzymes (NMPKs and NADK) as potential targets and different strategies have been used to identify inhibitors: structure-based drug design, in silicoand functional screening of chemical libraries, and fragment-based approaches.

UMPK from M. tuberculosis (UMPKmt) was characterized combining biochemical and structural investigations. The allosteric regulation of this enzyme was shown to be typical from Gram-positive bacterial UMPKs. The crystal structure of UMPKmt has enabled to define the positive effector binding site. Thanks tosite-directed mutagenesis, we have also pinpointed key residues involved in the « cross-talk » between the active site and the effector binding pocket.

Crystal structures of NADK from the human pathogen L. monocytogenesin complex with two natural ligands, NAD and NADP, as well as with synthetic NAD mimics were obtained. We described the first, 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 approach.

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 NMR structure of Rcl in complex with two different ligands was solved and the active site was probed by site-directed mutagenesis and by the use of substrate analogs. These studies provides the roadmap for the design of small molecules inhibitors with pharmacological properties.

Screening activity

The screening activity offers the possibility to identify small molecules as research tools or/and chemical starting points for therapeutic applications. The compound collection (around 42 000 compounds) comprises commercial and in-house synthesized molecules, and a part of the French academic chemical library ("Chimiothèque Nationale"). The offered resources consist of assay development and validation together with screening and data processing. Since the set up of the screening activity, 15 campaigns (enzymatic or whole-cell assays) have been performed in our research programs or by means of collaborative projects (3 patents). Hits in each project were validated in secondary assays and confirmed hits are currently under investigation in structure-activity relationship studies.

Keywords: Antibacterial Agents, Chemical library screening, Chemo-enzymatic approaches, Medicinal Chemistry, Nucleosides, Nucleoside metabolism enzymes

1. Dupouy, C.; Zhang, C.; Padilla, A.; Pochet, S.; Kaminski, P. A. (2010) Probing the Active Site of the Deoxynucleotide N-Hydrolase Rcl Encoded by the Rat Gene c6orf108, Journal of Biological Chemistry 285, 41806.

2. Labesse, G., Benkali, K., Salard-Arnaud, I., Gilles, A. M., and Munier-Lehmann, H. (2010) Structural and functional characterization of the Mycobacterium tuberculosisuridine monophosphate kinase: insights into the allosteric regulation, Nucleic Acids Research, doi:10.1093/nar/gkq1250.

3. Yang, Y.; Padilla, A.; Zhang, C.; Labesse, G.; Kaminski P.A. (2009) Structural characterization of the mammalian deoxynucleotide N-hydrolase Rcl and its stabilizing interactions with two inhibitors. Journal of Molecular Biology, 394, 435-47.

4. 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.

5. Caillat, C.; Topalis, D.; Agrofoglio, L. A.; Pochet, S.; Balzarini, J.; Deville-Bonne, D.; Meyer, P. (2008) Crystal structure of poxvirus thymidylate kinase: An unexpected dimerization has implications for antiviral therapy. Proc. Natl. Acad. Sci. USA, 105, 16900-16905.