EUR-INTAFAR - Inhibition of new targets for fighting antibiotic resistance

EC contribution: : € 11.301.842
Duration: : 60 months
Starting date: : 01/01/2005
Funding scheme: : Integrated Project
Keywords: : antibiotics, peptidoglycan, bacterial cell morphogenesis, transpeptidase, glycosyltransferase, penicillin-binding proteins, Lipid II, Mur proteins, Fem proteins
Contract/Grant agreement number: : LSHM-CT-2004-512138
Project web-site: : http://www.eur-intafar.eu

Background:

Peptidoglycan (PG) biosynthesis and bacterial cell morphogenesis are related phenomena and are totally specific to bacterial cells without even remotely equivalent systems in eukaryotic cells. The enzymes and proteins involved in these processes are thus promising targets for the design of new antibiotics. Interfering with the activities of the participating enzymes or with the protein-protein interactions that take place along these metabolic pathways should perturb the bacterial cell cycle and, hopefully, supply new weapons to fight dangerous pathogenic organisms such as the methicillin-resistant Staphylococcus aureus (MRSA).

Problem:

Although antibiotics have drastically reduced illness and death from infectious diseases, bacteria have exhibited a remarkable capacity to quickly become resistant to one or several classes of antibiotics. For example, in the US, until 2000, Streptococcus pneumoniae infections caused, each year, 100,000 to 135,000 hospitalisations for pneumonia, 6 million cases of otitis media and 60,000 cases of invasive diseases including 3,300 cases of meningitis. Up to 40% of the infections were caused by bacteria resistant to at least one and 15% to 3 or more antibiotics. The percentage of S. pneumoniae strains resistant to penicillin varies from 3.2 in The Netherlands to 53 in France. Values as high as 60 and 78% are observed in Hong Kong and Saudi Arabia, respectively. Resistance to ?-lactams is often associated to resistance to macrolides.
In 1996, 2 million cases of nosocomial infections were counted annually in US hospitals. Their global cost ranged from 600 $ for a urinary infection to 40,000 $ for a septicemia. Extrapolations made in 1996 in a French study showed that due to nosocomial infections, the stays in hospital were 3 to 7 days longer and the expenses per patient 750 to 1500 € higher. These annual extra costs represented about 2% of the total hospital expenses.
Strains isolated from farm animals present even higher levels of resistance. In a recent study performed in Belgium, 95% of the Escherichia. coli strains isolated from poultry, 44% of the strains of bovine origin and 90% strains of porcine origin were resistant to tetracyclines. In all of these cases, resistance to aminoglycosides (streptomycin), chloramphenicol and amoxycillin or ampicillin was also widespread. The increase in antibiotic resistance is thus a global problem, both for nosocomial as well as community-acquired infections. A return to the “pre-antibiotic” era has even been forecasted. Hence the problem of resistance can only be solved by a multidisciplinary and international approach, which will require a better understanding of the fundamental aspects of bacterial physiology, growth and multiplication mechanisms, areas which have been relatively neglected in the recent past when compared to eukaryotic systems.

Aim:

The aim of this network is to find new targets for antibiotics and to use the knowledge accumulated on the antibiotic-resistant forms of the “old” targets for the design of more efficient molecules.
Peptidoglycan (PG) biosynthesis requires reactions catalysed by soluble intracellular enzymes followed by a series of steps involving membrane-bound proteins during which the disaccharide-peptide moiety is assembled, transported across the cytoplasmic membrane and finally polymerised and cross-linked in the periplasmic or outer space by transglycosylation and transpeptidation reactions. Moreover, to allow cell growth and division, the PG network must be continuously remodelled thanks to the additional participation of hydrolases. Cell elongation and division are thus closely related to PG biosynthesis and metabolism and all the proteins/enzymes involved in these highly specific phenomena are potential targets for new antibacterial compounds.

Expected and obtained results:

Transpeptidases (Tpases) or Penicillin-Binding-Proteins (PBPs) are the targets of ß-lactam antibiotics. However, some pathogenic bacteria such as Streptococcus pneumoniae and the methicillin-resistant Staphylococcus aureus MRSA have acquired transpeptidases that are resistant to most clinically useful ß-lactams. Various novel methods have been devised to test inhibitors prepared by the chemist partners. In an effort to identify compounds which do not contain a ß-lactam ring but inactivate PBPs, we have shown that lactivicin, the only known natural compound exhibiting such properties is active against clinically isolated penicillin-resistant S. pneumoniae strains. Crystallographic studies performed with S. pneumoniae PBP 1b reveal that the inactivation reaction involves opening of both cycloserine and ß-lactone rings of lactivicin (see illustration below). Thus, lactivicin derivatives will be useful in the search for antibiotics active against ß-lactam resistant bacteria.
Publication:
“Structural and mechanistic basis of penicillin-binding protein inhibition by lactivicins”
P. Macheboeuf, D. Fischer, T. Brown Jr, A. Zervosen, A. Luxen, B. Joris, A. Dessen and C. Schofield
Nature Chemical Biology Letters. 3, (9) 2007, 565-569

Crystal structure of Streptococcus pneumoniae penicillin-binding protein 1b (PBP1b). On the left: PBP1b-nitrocefin complex; on the right: PBP1b-phenoxyacetyl-lactivicin (PLTV) complex.

Other PBP and PBP-inhibitor structures have also been solved. Surprisingly, we showed that ß-lactam resistance in S. pneumoniae also requires the CiaRH regulatory system, which is not directly related to PG biosynthesis.
Publication:
“The CiaRH system of Streptococcus pneumoniae prevents lysis during stress induced by treatment with cell wall inhibitors and mutations in pbp2x involved in beta-lactam resistance”
T. Mascher, M. Heintz, D. Zähner, M. Merai and R. Hakenbeck
J. Bacteriol. 188, (5) 2006, 1959-1968
No clinically useful inhibitor of the glycosyltransferase (GTase) activity is known . Several monofunctional GTases were purified as well as two class A PBPs exhibiting dual Tpase GTase activities. On the basis of published GTase structures, a virtual screening of small molecules from the NCI Diversity set was performed, yielding 30 potential inhibitors among which two were found to inhibit the GTase activity of E.coli PBP 1b. Studies of the GTase-catalysed reactions are often impaired by the fact that the only known substrate is the complex, water insoluble, lipid II. A new method has been devised to prepare relatively large amounts of purified lipid II in a reproducible manner.
The steps preceding transglycosylation and transpeptidation result in an outward-oriented lipid II. They involve the synthesis of lipid II by MraY and MurG followed by translocation of the disaccharide-peptide moiety across the cytoplasmic membrane. The MurG and MraY proteins have been purified to homogeneity and characterized. In the search for inhibitors/inactivators, some of the difficulties inherent to the synthesis of the complex substrate analogues have been solved.
Publication:
“Efficient synthesis of polyfunctionalized enantiopure diazepanones scaffolds”
O. Monasson, M. Ginisty, G. Bertho, C. Gravier-Pelletier and Y. Le Merrer
Tetrahedron 48, 2007, 8149-8152

Assays which monitor the transport of the disaccharide-peptide moiety across the membranes have been set up and used in combination with photo crosslinking approaches to identify the proteins participating in this process.
Publication
“Transmembrane transport of peptidoglycan sub-units across model and bacterial membranes”
V. van Dam, R. Sijbrandi, M. Kol, E. Swiezewska, B. de Kruijff and E. Breukink
Mol. Microbiol, 64, 2007, 1105-1114
Only two clinically useful antibiotics are presently available which target the intracellular steps leading to the soluble PG precursors and one of them (cycloserine) might be withdrawn in the near future. All the intermediate metabolites have been prepared and a novel class of MurD inhibitors has been identified. The structure of an enzyme inhibitor complex has been solved.
Publication
“Structural and functional characterization of enantiomeric glutamic acid derivatives as potential transition state analogue inhibitors of MurD ligase”
M. Kotnik, J. Humljan, C. Contreras-Martel, M. Oblak, K. Kristan, M. Hervé, D. Blanot, U. Urleb, S. Gobec, A. Dessen and T. Solmajer
J. Mol. Biol., 370, 2007, 107-115

In Gram-positive bacteria, the Fem transferases are attractive targets because of their essential role in ß-lactam resistance and unique mode of action which involves aminoacyl-tRNAs. An analogue of this substrate that inhibits Fem X with an ICso of 0.17 µM has been synthesised.
Publication
“Stable analogues of aminoacyl-tRNA for inhibition of an essential step of bacterial cell wall synthesis”
M. Chemama, M. Fonvielle, R. Villet, M. Arthur, J.M. Valéry and M. Etheve-Quelquejeu
J. Am. Chem. Soc., 129, 2007, 12642-12643
It is now widely accepted that cell elongation and septation involve dynamic supramolecular assemblages containing a large number of proteins including those which catalyse the last steps of PG synthesis. On the basis of a newly developed FRET assay and other efficient techniques, we have shown that septal PG is synthesised by a complex containing PG synthases and hydrolases whose functions are controlled by other cell division proteins. In E .coli, the MurG protein forms complexes both with proteins involved in lateral growth as well as in septation. In particular, the existence of complexes containing PBP1b and PBP3 has been demonstrated and the elongasome responsible for lateral growth consists of MreB, MraY, MurG, MreC, MreD, RodA, PBP2 and PBP 1a. In S. pneumoniae, new enzymes participating in teichoic acid biosynthesis have been identified and the long-standing problem about why S. pneumoniae requires choline for growth was solved.
Publications:
“Interaction between two murein (peptidoglycan) synthases, PBP3 and PBP 1b in Escherichia coli”
U. Bertsche, T. Kast, B. Wolf, C. Fraipont, M.E.G. Aarsman, K. Kannenberg, M. von Rechenberg, M. Nguyen-Distèche, T. den Blaauwen, J.V. Höltje and W. Vollmer
Mol. Microbiol., 61, 2006, 675-690

“The essential peptidoglycan glycosyltransferases MurG forms a complex with proteins involved in lateral envelope growth as well as with proteins involved in cell division in Escherichia coli”
T. Mohammadi, A. Karczmarek, M. Courvoisier, A. Bouhss, D. Mengin-Lecreulx and T. den Blaauwen
Mol. Microbiol., 65, 2007, 1106-1121
An innovative high-throughput system, has been developed for screening chemical compound libraries in microspots (EU patent application submitted, February 2008).
Up to 30/04/2008, the network had produced a total of 60 publications in highly regarded journals

Potential applications:

New antibiotics are needed to fight multi-resistant pathogens. Large pharmaceutical companies led the field of antibacterial research for a number of years but have failed to deliver new antibiotics in recent times and some of them exhibit significantly decreased interest for this area. Although the potential antibiotic market remains huge, up to 90% of infections can be treated with the presently available compounds. The design of antibacterial agents directed towards specific species or strains is a sensible strategy from a public health point of view. It is much less interesting commercially. Indeed, a good antibiotic is expected to be taken by the patient over a short period of 1-2 weeks. In consequence, if public authorities do no take charge of the problem, it will remain unsolved at a large cost for the society.
This project is of prime importance as a springboard to re-activate the important therapeutic area of antibiotic drugs.
A better understanding of the physiology and biochemistry of bacterial cell morphogenesis and peptidoglycan biosynthesis will create new avenues for the design and synthesis of efficient antimicrobials. This will make new opportunities available for companies of different sizes to develop these compounds until they reach the clinical level.

Coordinator:

Prof. Jean-Marie Frère
Centre for Protein Engineering
Institut de Chimie B6a
University of Liège
Sart-Tilman
B400 Liège, Belgium
Tel 32 4 366 33 98
Fax 32 4 366 33 64
Email:jmfrere@ulg.ac.be

Graphical representation of the Eur-Intafar integrated project

The global structure of the Eur-Intafar project involves five workpackages, addressing individual reactions (WPs 1 and 2), parts of a biosynthetic pathway (WPs 3 and 4, divided according to the cellular location of the enzymes) and a complex cellular process and its control (WP5). The latter WP also aims at integrating the data obtained in the others, so that a general but detailed picture of cell growth and division will be obtained. In workpackages 3, 4 and 5, specific reactions or interactions will be identified as especially amenable to interference by an inhibitor which might become a lead compound in the search for new antibiotics. The workpackages are subject-oriented.

The management (see below) is headed by the coordinator who is assisted by the project manager, the scientific managers and workpackage leaders.

European Commission