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Development of new antibacterial and antiparasitic drugs target of the GcpE metalloenzyme – ANTIBIO-T

The design of new drugs by blocking an enzyme

Elucidation and inhibition of the catalytic mechanism of GcpE, an enzyme absent in humans but present in many bacteria and some parasites.

A new solution to overcome microbes resistant to antibiotic drug therapies

The emergence and spread of antimicrobial resistance is a serious threat. The development of new drugs acting on new targets is timely. Therefore the elucidation of so far unknown biochemical pathways which are indispensable for the survival of pathogenic bacteria could be a solution to this problem. The gathered knowledge of such pathways and the corresponding reaction mechanisms of the enzymes involved can help chemists and biochemists to develop inhibitory compounds which are candidates for new antimicrobial compounds.<br /><br />The methylerythritol phosphate pathway (MEP) is used for the biosynthesis of compounds (called ‘terpenoids’) indispensable for the survival of most pathogenic bacteria (including Mycobacterium tuberculosis responsible for tuberculosis), opportunistic (Pseudomonas spp, Escherichia coli…), the parasite responsible for malaria. This pathway does not exist in humans and therefore it could be considered to block this pathway in order to develop new specific antibacterial and antiparasitic drugs.<br /><br />The aim of this project is to elucidate the mechanism catalyzed by GcpE, the penultimate enzyme of the MEP pathway, and to design ‘inhibitors’ that are molecules capable of blocking this enzyme.<br /> GcpE is a metalloenzyme containing an iron/sulphur center, that means a cube having an iron atom and a sulfur atom alternately on each vertex. This enzyme catalyses through its iron/sulphur center two one-electron transfers and the elimination of a water molecule. A hypothetical reaction mechanism has been written that we propose to verify. Molecules capable of preventing the functioning of this mechanism will also be synthesized.

Molecules with analogy to the substrate of GcpE will be synthesized and their effect on GcpE will be tested. As GcpE is oxygen sensitive, these tests are performed in a special device called 'glove box' that ensures the stabilization of the amount of oxygen to 2 ppm maximum. The interaction of these molecules with the iron/sulfur center of GcpE will be verified by using different spectroscopies such as, for example Mössbauer spectroscopy that is specific for iron. The X-ray diffraction will provide access to the three-dimensional structure of GcpE in the presence of these molecules.

The results will shed light onto the mechanism of action of GcpE, and the acquired knowledge will be used to design new drugs.

The requirements to carry out this project are the obtention of the GcpE enzyme in a soluble, stable, homogeneous, non-aggregated form and in large amount (several milligrams), and the availability of the molecules to be tested.

A lock of this project was the obtention of pure GcpE containing a [4Fe-4S] per molecule of protein. The GcpE enzyme of Escherichia coli has been cloned and we have developped a method for the production of this enzyme in large quantities containing a Fe /S center per molecule.
Some GcpE substrate analogues are being currently synthesized. A method for the synthesis of the substrate has been validated.

A protocol for expression and purification of the GcpE enzyme of another bacterium has been developed. The first crystallogenesis trials performed with a crystallization robot located inside the glove box led to the first GcpE crystals. One of these crystals allowed the resolution of the three-dimensional structure of a homologous of GcpE from E. coli.
Several GcpE mutants were prepared by site-directed mutagenesis. The evaluation of their activity is on going.

The most active inhibitors of GcpE will be tested on bacteria to check if they reduce their viability. Clinical trials will then follow and perhaps the placing on the market of a new drug will result.

The results of this work were presented as oral presentations. They will also be published.

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.

Project coordinator

Madame Myriam Seemann (UNIVERSITE DE STRASBOURG) –

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.



Help of the ANR 459,999 euros
Beginning and duration of the scientific project: February 2012 - 48 Months

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