With the emergence of antibiotic resistance worldwide, the search for new antibacterial compounds has become increasingly important. With the aim to identify inhibitors based on novel mechanisms of action, we selected key bacterial enzymes as potential targets, and applied different strategies (structure-based drug design, in silico screening, functional screening of academic and commercial libraries, fragment-based approaches) to define inhibitors.
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. Our research projects have been focused on NMPKs of bacterial pathogens, mainly on M. tuberculosis responsible for tuberculosis (collaboration with S. Cole and G. Marchal, Institut Pasteur) and on their human counterparts (collaboration with D. Deville-Bonne, Paris VI).
The five NMPKs of M. tuberculosis have been studied by various biochemical and physico-chemical techniques (Munier-Lehmann et al. 1999, 2001; Hible et al., 2006). The 3D-structures of TMPK (collaboration with M. Delarue, Institut Pasteur, and D. Bourgeois, Grenoble) (Li de la Sierra et al., 2001; Fioravanti et al. 2003, 2005), GMPK (collaboration with J. Cherfils, Gif sur Yvette) (Hible et al., 2006) and AMPK (collaboration with G. Craescu, Orsay) (Miron et al. 2001, 2004), have been solved in apo or substrate-bound forms. These studies have revealed that these NMPKs have intermediate properties between eukaryotes and prokaryotes and peculiar structural features making them potential targets for the design of new antitubercular drugs.
Our synthetic efforts were mainly devoted to the search of potent inhibitors of TMPK of M. tuberculosis (TMPKmt). This protein was also used to develop and validate bioinformatics programs in collaboration with the CBS in Montpellier (Douguet et al. 2005). Based on the 3D-structure of TMPKmt in complex with dTMP, one of its natural substrate, we have started a drug design program, as well as the in silico screening of chemical libraries. Different classes of molecules have been synthesized as substrate mimics: thymidine analogues, benzyl-thymine derivatives predicted by a drug design program (LEA3D), and bisubstrate inhibitors. The inhibitory efficiency of the synthesized compounds was determined in vitro on the recombinant enzyme using a coupled spectrophotometric assay. The compounds exhibiting the highest affinities were tested on the human TMPK for specificity measurement. We have thus identified key residues governing the substrate specificity of TMPKmt (Munier-Lehmann et al. 2001; Pochet et al. 2002, 2003b) and identified original molecules acting in vitro as selective inhibitors of TMPKmt (Pochet et al. 2003; Vanheusden et al. 2002, 2003a, 2003b, 2004; Familiar et al., 2008) and on mycobacteria strains (Van Daele et al. 2006, 2007; Gasse et al. 2007, 2008). The most potent compounds against TMPKmt have Ki values in the micromolar range and an MIC50 of 7 µg/ml against M. bovis (BCG) and H37Rv strains. These molecules could represent a new class of antituberculosis agents (in collaboration with S. Van Calenbergh, Belgium and M.J. Perez-Perez, Spain). We are currently working on further modifications and optimizations.
A versatile automated platform (TECAN Freedom EVO® platform) with open-architecture software capable of integrating off-the-shelf peripherals was set up in 2007. This workstation is housed within a biosafety level 2 room and is placed under laminar flow hoods. It includes: (i) temperature and CO2 controlled storage units, (ii) robotic manipulator arms, (iii) high precision liquid handling arms with 4 and 8-channels using washable or diposable tips, (iv) Te-MO 96-channel pipetting heads, (v) bar code readers, (vi), labware storage carousel and carriers (vi) shakers with peltier modules, (viii) microplate readers (SunriseTM and Safire2TM) for absorbance, fluorescence and luminescence measurements, all running under the control of FACTS™ (Flexible Assay Composer and Task Scheduler) software. The data are stored, processed and analyzed through a LIMS configured and maintained by Modul-Bio.
Diverse chemical libraries (around 40 000 compounds) including the French National Chemical Library (“Chimiothèque Nationale”), commercial and in-house libraries are screened on purified enzymes from our laboratory and on cellular assays (in collaboration with partners from Institut Pasteur). Seven campaigns were performed in 2009 covering various biological areas in anti-infectious and anti-cancer domains, leading to two patents and validated hits in secondary assays.