CombiGyrase - Development of new gyrase inhibitors by combinatorial biosynthesis

EC contribution: : € 1.555.415
Duration: : 36 months
Starting date: : 01/01/2004
Funding scheme: : Specific Targeted Research Project
Keywords: : antibiotics, resistance, anti-infective, gyrase, aminocoumarin, simocyclionone
Contract/Grant agreement number: : 503466
Project web-site: : -

Background:

The bacterial enzyme DNA gyrase is well validated as a target for a number of antibacterial compounds. The CombiGyrase consortium took advantage of the expertise that existed across Europe to research and develop new drugs that are urgently needed. It represented an ideal platform to expand the diversity of potent gyrase inhibitors found in nature by methods of combinatorial biosynthesis. Combinatorial biosynthesis is a novel technology that uses genetic manipulation to improve the chemical properties and pharmacological activity of naturally occurring compounds. Using microorganisms which produce natural gyrase-inhibiting antibiotics, the CombiGyrase consortium successfully demonstrated that novel 'designer' antibiotics can be developed by combinatorial genetic methods. New gyrase-directed drugs, such as aminocoumarin and simocyclinone antibiotics, developed by these methods, may help to overcome problems due to clinical resistance, and may significantly expand the clinical role of the gyrase inhibitors as antibacterial agents.

Problem:

A constant threat to the population of the European Community is the ever-increasing problem of antibiotic resistance. Widespread use of antibiotics has led to the emergence of antibiotic-resistant strains. The increase and spread of resistance are a matter of serious public health concern worldwide. For example, vancomycin has long been considered as the solution to methicillin-resistant Staphylococcus aureus (MRSA) infections, but vancomycin-resistant strains of S. aureus have already begun to emerge. Nowadays, the risk of infection increases with a prolonged hospital stay, and so does failure of antibiotic therapy because of multidrug resistance.

In the last decade, many pharmaceutical companies have reduced their efforts to discover new anti-infectives, particularly new antibiotics, and redirected their R&D efforts to other fields perceived as more profitable. However, infectious diseases represent a large market with high unmet medical needs, as shown by the strong interest of many large pharmaceutical companies to acquire novel antibiotics once they have reached late development/registration stages.

Aim:

The principal objectives of the CombiGyrase project were:

the development of new antiinfectives, that target gyrase and/or bacterial topoisomerase IV, by combinatorial biosynthesis in Streptomycetes;
the evaluation of the activity of these compounds as inhibitors of gyrase and of topoisomerase IV, and of the resulting antibacterial activity against bacterial pathogens;
the evaluation of the suitability of these compounds as drug candidates, especially for the treatment of infections with multi-drug-resistant bacteria.

The focus was on the development of derivatives of the following antibiotics, which are produced by different Streptomyces strains and represent highly potent inhibitors of gyrase:

the aminocoumarin antibiotics novobiocin, clorobiocin and coumermycin A1;
the mixed aminocoumarin/angucycline antibiotic simocyclinone D8;
the mixed peptide/polyketide antibiotics cyclothialidine and GR122222X.

Expected and obtained results:

Using microorganisms which produce natural gyrase-inhibiting antibiotics, the CombiGyrase consortium successfully demonstrated that novel 'designer' antibiotics can be developed by combinatorial genetic methods.

A model showing how the antibiotic novobiocin (in blue) is bound to its target DNA gyrase (yellow ribbon) and how it prevents binding of the substrate molecule (ATP, in red)

Novobiocin and clorobiocin are aminocoumarin antibiotics and potent inhibitors of bacterial gyrase. Structurally, the two compounds differ by the substitution pattern at two positions. Using genetic engineering of the antibiotic-producing microorganisms, we generated a series of structural analogs of these antibiotics. In a subsequent cooperative study by two partners of the CombiGyrase consortium, comparison of the inhibitory activity of these compounds on gyrase and on topoisomerase IV provided the first systematic evaluation of aminocoumarins against both drug targets, and provided new, detailed insight into the structure-activity relationships of these antibiotics. This creates the basis to design more potent agents of antiinfective drugs from this class.

Utilizing the genetic, biotechnological, bioinformatic and pharmacological knowledge generated in the CombiGyrase consortium, 33 new aminocoumarins were generated by mutasynthesis experiments using genetically optimized microorganisms. The structures of these compounds were elucidated, and in cooperation between three partners of the consortium, the new antibiotics were tested for gyrase inhibition in vitro and in a cell-based reporter gene expression assay, and for their activity against bacterial pathogens. A high-throughput assay for the ATPase activity of gyrase B was established and validated with known inhibitors. This assay was used to screen >30 novel novobiocin analogues. Several compounds with anti-microbial activity in the same range as the most potent known antibiotic, clorobiocin, were identified. A secondary assay for detecting the mode of action of novel anti-microbial compounds, based on the induction of target promoters at sub-inhibitory concentrations of antibiotics, was validated for gyrase B inhibitors. The mode of action of the novobiocin analogues on gyrase B was confirmed. Routine cytotoxicity testing was used to further characterize the new derivatives. Cytotoxicity testing using several cell lines was also used to profile a series on modified coumarins that had potential to be topoisomerase II inhibitors.

We discovered the mode of action of a completely novel class of DNA gyrase inhibitors (the simocyclinones). Simocyclinones share some structural similarities with aminocoumarins but a number of differences. We have found that these compounds target gyrase and that simocyclinone D8 is a more potent inhibitor than novobiocin. Surprisingly simocyclinones inhibit gyrase by a mechanism that is distinct from that of the aminocoumarins; rather than binding at the ATPase site of GyrB they bind to GyrA and prevent DNA binding. This is a completely novel mechanism of gyrase inhibition and is of great interest in terms of the development of new anti-bacterials.

To obtain information on the drug-protein contacts we now crystallized the N-terminal fragment of GyrA (GyrA59) in the presence of simocyclinone D8. This has yielded crystals that diffract to ~2.6 Å and we have now solved the structure. The mode of binding of the drug is completely novel and is consistent with binding measurements using the Biacore T100.

(a) (b)

Crystallography of the GyrA59-simocyclinone D8 complex: (a) large single crystals grown by hanging drop vapour diffusion; and (b) preliminary X-ray diffraction analysis.

Potential applications:

The CombiGyrase project was designed to improve European competitiveness in the promising new technology of drug development by combinatorial biosynthesis. The results benefit public health by providing a road to new antibiotics which will help to combat infectious diseases. New generations of gyrase-directed drugs, such as aminocoumarin and simocyclinone antibiotics, developed by methods of combinatorial biosynthesis and targeting gyrase and/or topoisomerase IV, will help to overcome problems due to clinical resistance, and may significantly expand the clinical role of the gyrase inhibitors as antibacterial agents.

Coordinator:

Prof. Dr Lutz Heide
University of Tübingen
Pharmazeutisches Institut
Auf der Morgenstelle 8
72076 Tübingen, Germany
Tel. +49 (07071) 297 2460
Fax +49 (07071) 295 250
heide@uni-tuebingen.de

Participants of the CombiGyrase consortium at a project workshop in Basel, Switzerland, Feb 2005

European Commission