Bacteria adapt quickly to their environment and also to antibiotics. Many of the antibiotics used to treat bacterial infections have become ineffective as a great many bacteria have become resistant to them. Freiburg-based FreiBiotics GmbH is looking for completely new classes of antimicrobial substances. A screening method that has been developed over the last few years based on biosensors makes the identification of new substance classes more efficient at the same time as reducing the costs associated with their development. This screening method not only provides information about the efficiency of a potential antibiotic, but also about the underlying molecular mechanisms. FreiBiotics GmbH is ready for market entry with this new technology and is looking for industrial cooperation partners.
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An increasing number of bacterial pathogens are becoming resistant to drugs and, at the same time, there is a continued decline in the number of newly approved drugs. A global public health crisis is therefore becoming increasingly likely. Infectious diseases are the second most frequent cause of death around the world. Multidrug resistant pathogens, i.e. pathogens that are resistant to more than just one antibiotic, are the greatest problem. Hospitalised patients or elderly people with deficient immune systems are particularly prone to bacterial infections for which effective antibiotic therapies are no longer available. “New classes of antibiotics are urgently needed,” said Dr. Christian Schleberger, head of bioinformatics at FreiBiotics GmbH in Freiburg. “We are offering a new technology that enables the efficient search for effective drugs, which is both more specific and cheaper than previous methods.”
FreiBiotics was spun out of the University of Freiburg in 2009 and is located in the rooms of the Department of Pharmaceutical Biology and Biotechnology, which is run by Prof. Dr. Andreas Bechthold. The know-how associated with this new method, which will be marketed worldwide, is based on the work of Prof. Dr. Dirk Bumann carried out as part of the GO-Bio project funded the German Ministry of Education and Research (BMBF). Bumann, who is one of the founders of FreiBiotics and visiting professor at the University of Freiburg, now works at the Basle-based Biocentre (Switzerland). On the basis of his work involving bacterial pathogens such as Salmonella, Bumann developed the principle of biosensor-assisted drug screening known as Bumann’s Antibacterial Screening Platform (BASP). FreiBiotics is particularly focused on the search for drugs that combat Gram-negative bacteria, which are characterised by a double cell wall. E. coli is one such Gram-negative bacterium. In 2010, the five members of the FreiBiotics team optimised and validated the method and are now ready to test potentially antimicrobial substances.BASP has numerous advantages over previous screening methods. It works on the basis of genetically modified E. coli bacteria. The researchers have incorporated a gene for a green fluorescent protein (GFP) variant into the genomes of E. coli cells. Every gene coding for a particular colour was combined with a promoter, a DNA sequence located near the gene it regulates (on the same strand and upstream) and that initiates the transcription of this particular gene. When a cell wants to produce a certain protein, the first thing it needs to do is to activate the promoter of a particular gene. The promoters used in the BASP method were selected by Bumann’s team using a process that was specifically developed for this purpose. The promoters can be activated by specific inhibitors of key cellular processes such as cell wall assembly and protein biosynthesis, but not by unspecific substances that would just lead to the death of the cell.
“A genetic construct of this kind is a biosensor for a substance that inhibits specific biochemical processes in bacterial cells,” said Schleberger going on to add “if we introduce an unknown substance into the cell, for example a substance that induces the assembly of the cell wall, then this substance activates the corresponding promoter, which in turn results in the transcription of the GFP gene downstream of the promoter. The cell can be visualised under the fluorescence microscope due to the specific colour of the GFP gene that has been inserted downstream of the promoter.” The researchers use a cytometer to measure the bacterial cells that irradiate in different colours. Subsequent computer analyses then provide information about which test substance has which effect. This method does not merely detect substances that kill bacteria. It detects substances that inhibit a specific process in the bacterial cell. This is one of the financial advantages of the technology: Up until now, pharmaceutical companies have tested their candidate molecules in cell cultures without looking into the bacterial cell. A potential drug became interesting if it led to the death of bacteria. Huge sums of money were then invested into further research and in many cases the companies realised far too late that the selected substance was unspecific and hence unsuitable for the intended purpose.
Another advantage becomes obvious when comparing BASP with currently used test methods that target a specific inhibitory mechanism. In principle, it is possible to investigate potential drugs in enzyme assays, in which substances of interest are brought together in a test tube with an enzyme involved in bacterial cell wall synthesis. The question to be answered is, which substance is inhibited by this particular enzyme? Although this is a rather specific test, it does not take into account an important aspect of the drug discovery process: how does the potential drug get into the bacterial cell? “This also requires numerous investigations, which cost a lot of money and which lead to the exclusion of numerous substances that have been identified as potential candidates,” said Schleberger. “Our method only identifies substances that have already entered the cell. Therefore, our method combines the advantages of unspecific test methods on the basis of living bacteria with those of specific enzyme assays.”
FreiBiotics can screen around twelve thousand molecules per month. “This is not much in comparison to other methods,” said Schleberger. “However, when you take into consideration the fact that our method enables us to make statements on a potential mechanism of action, the situation looks completely different: We do not require the large number of experiments that other methods do, and our method costs only around one tenth of a classical screening method.” The company hopes to double the current screening rate during the second phase of the GO-Bio project. In addition, the FreiBiotics team hopes to develop further biosensors with an even greater screening selectivity.After the initial phase of method development, FreiBiotics is now looking for industrial cooperation partners who are prepared to support the company in its effort to identify interesting drugs. The company does not want to be just a service provider that only provides its know-how to its clients. “We have the ambitious goal to find a potential drug and take it to clinical testing,” said Schleberger explaining that his company has already identified two promising candidates that are now being investigated further.
Further information:Dr. Christian SchlebergerR&D BioinformaticsFreiBiotics GmbHStefan-Meier-Str. 19 D-79104 Freiburg Tel.: +49 761 203 6240Fax: +49 761 203 6422E-mail: Christian.Schleberger(at)freibiotics.com