ANTIBIO-T

The methylerythritol phosphate pathway (MEP) is used for the biosynthesis of essential terpenoids in most pathogenic bacteria (including Mycobacterium tuberculosis responsible for tuberculosis), opportunistic (Pseudomonas spp, Citrobacter spp, Escherichia coli…), Plasmodium falciparum the parasite involved in malaria and in plant plastids. This pathway does not exist in humans and is therefore a target for new antibacterial and antiparasitic drugs. Fosmidomycin, a natural antibiotic, was shown to be an effective inhibitor of 1-deoxy-D-xylulose 5-phosphate synthase, the first enzyme of the MEP pathway. Patients with uncomplicated P. falciparum malaria were successfully treated with fosmidomycin but in some cases the parasite reappeared after the treatment. This first result shows the promising potentiality of the MEP pathway enzymes as drug targets and indicates the need for further improved inhibitors. One way to improve efficacy is to use a drug combination. This could be achieved by blocking several enzymes of the MEP pathway. The penultimate step of the MEP pathway is catalysed by GcpE, a metalloenzyme containing a highly oxygen sensitive [4Fe-4S] centre. The ultimate goal of this proposal is to elucidate the reaction mechanism of GcpE, and use the acquired knowledge to design new drugs. The reaction catalysed by GcpE involves two one-electron transfers and generate radical intermediates. According to the proposed mechanism, an interaction between an iron atom of the cluster [4Fe-4S] and the substrate may exist. Substrate analogues will be synthesised and used as molecular tools in the elucidation of the mechanisms. After evaluation of the potential of inhibition of these analogues on GcpE, EPR, Mössbauer spectroscopy experiments will be conducted to try to identify the radical intermediates and to investigate the mode of binding of these substrate analogues with the active-site [4Fe-4S] cluster. Complementary information on the mechanism of GcpE will be obtained by FTIR difference spectroscopy, Raman and electrochemistry. In order to get a better insight into the active sites, we will attempt to solve the crystal structure of GcpE in complex with the substrate or substrate analogues. The importance of the residues revealed by the X-ray structure will then be confirmed using site-directed mutagenesis. Several point mutated proteins will be constructed. The biochemical characteristics (steady-state kinetic parameters) of each mutant will then be determinate in order to see if the mutation is effective. The three dimensional structure will be used as a target for screening virtually chemical libraries in order to access to the first in silico drugs. These molecules will then be synthesized and tested in vitro (SAR studies). Finally, we propose to realise a high throughput screening (HTS) on the MEP pathway using the Prestwick Chemical Library (1200 off-patent drugs) together with a modified E. coli strain in order to identify new antibacterial and antiparasitic drugs.