We propose an inter-disciplinary research and training programme focusing on understanding the molecular basis of an important antibiotic resistance mechanism. Recent advances in biophysical methods make it possible to study the relationship between permeation of antibiotics into the cell and bacterial resistance. This proposal makes use of recently developed biophysical techniques bridging the gap between biophysics and microbiology in order to improve our understanding of the contribution that restricted permeation makes to bacterial resistance. A special strength is the participation of two strong industrial partners that will ensure the transfer of basic knowledge from academia to industry and apply it to the design of new antibiotics and screening technologies. In addition to answers to an exciting scientific question we expect a number of well-trained students with a good chance on the job market and a new screening technology for channel related drugs.
          Beta-lactam antibiotics are commonly used for the treatment of bacterial infections(1-6).Their target is the biosynthesis of peptidoglycan, the major component of the bacterial cell wall. The beta-lactams inhibit the biosynthetic enzymes triggering a complex series of events that leads to lysis and death of the bacteria. Gram-negative bacteria, which account for around half of the life-threatening infections in clinics today, have an outer membrane that helps to protect the cell by limiting the entry of even quite small molecules. In order to cross the outer membrane, the beta-lactams must pass through water-filled channels formed by special proteins - porins - located in the outer membrane. Beta-lactam-resistant strains of pathogenic Gram-negative bacteria frequently have a deficiency in the expression of the general diffusion porin OmpF (Outer membrane protein F) that leads to resistance (3,4). Further, alterations of porins or production of porins exhibiting narrow channels (6) (decreased pore radius) have been shown to strongly inhibit antibiotic uptake (4). These studies show the vulnerability of bacteria towards these agents and imply that beta -lactam antibiotics permeate into the periplasm through OmpF channels. Counteracting bacterial resistance to penicillins and cephalosporins requires a better knowledge of the molecular basis governing beta-lactam penetration across the outer cell wall via porins.

          In 2002 one of our teams was able to improve the signal to noise ratio in planar bilayer measurements to the point where it was possible to follow single antibiotic molecules along their pathway to the target (7,8). Such high-resolution measurements allow questions regarding possible mechanism, pathways and means of inhibition to be addressed directly. For example, the detection of binding sites for drugs inside the channel facilitating the translocation across the outer bacterial membrane has become feasible through these developments (7-9). In addition, powerful new computers and algorithms enable us now to combine this information with high-resolution protein structures to identify potential binding sites and to suggest modifications of drugs in order to improve its translocation through the outer membrane channels (9,10). Experiments have pointed out that the translocation process is essentially governed by molecular properties. Combining the high-resolution protein structures with accurate molecular modelling makes it possible to investigate the mechanism at atomic level in detail (10). Molecular modelling provides detailed information on a given process with high resolution in both time (femtosecond) and space (Ångstroem). However, biological processes run on time scales of microseconds, well beyond typical times of computer simulations. Recent advances in algorithms and computers have extended simulation times and system sizes to scales very close to the ones characterizing these biological processes without loosing accuracy (11,12). Successful examples of coupling experimental data to numerical simulations have been seen during the investigation of the binding of small molecules to proteins (9,13). We expect that such research procedures will allow to identify potential binding sites of antibiotics inside the channels and to suggest modifications of drugs to improve their translocation.

          These recent technological advances make it worth revisiting the problem of drug translocation across the bacterial outer membrane. Although the current state of this technology enables high resolution to be attained, the techniques are very time consuming. Here, the application of new advances in micro-fluidics can facilitate automation and miniaturization that will be essential for the technique´s use in industrial drug discovery (14,15). Since 2003-2004, the first feasible solution for high-throughput screening of membrane channels was proposed. The consortium includes Nanion, one of the leading players in this field, as we plan to scale-up our test to allow rapid screening of large numbers of potential active molecules (16,17). We will design and develop a completely new micro device and test its performance. Basilea, a first-class industrial player in research and development of antibiotics completes the consortium. Close connection with a partner from the pharmaceutical industry will give the other members of the consortium access to a large number of unpublished compounds and to relevant information concerning antibiotic resistance.

          As outlined above, the initial proof of principle of the approach has been achieved. What is now needed is to put this hypothesis on a solid scientific basis by a larger series of experiments to see the power and the limits of this approach. For this we need a larger consortium of broad expertise to guarantee a rapid outcome. In close collaboration with two SMEs we will investigate in parallel the feasibility of our technology. We expect that this combined integrated approach, ranging from biophysics to clinical microbiology provides new insight in antibacterial research, yields new antibacterial drugs and, more generally, delivers new methods for screening approaches of membrane channels.

References
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