New tool for characterising antibiotic-target binding
At present there are very few truly new anti- biotics in the global drug development pipelines. This is a continual source of concern for healthcare professionals as there is an increasing prevalence of multidrug resistant (MDR) healthcare acquired infections (HCAIs), which are also now permeating beyond the bounds of healthcare settings. The MDR pathogens are, on the whole, mutated strains of common bacteria or fungi. For example Staphylococcus aureus is a common coloniser of human skin and its native strain causes very few problems, but mutant strains resistant to many antibiotics (commonly referred to as methicillin resistant S. aureaus [MRSA]) have a high morbidity and mortality. Similarly, Enterococci spp. (e.g. E. faecalis and E. faecium) are commensal organisms in the digestive tract, but highly virulent strains resistant to virtually all antibiotics have emerged.
Bending the Rules
Developing new antibiotics capable of treating these resistant strains is therefore a key priority, but there is a requirement for new tools to make the drug discovery process more efficient. An important bottleneck to widen is the characterisation of the interactions between lead compounds and their targets. Here, a novel drug-target binding detection system is proving itself to be a big hit. Based on micro-cantilever technology it provides exquisite information on the nature of a drug-target interaction and can even discriminate between different antibiotics. Furthermore, it can be applied on a high throughput basis making the search for new products much quicker too.
The cantilever approach
Micro-cantilevers have been shown to be essential to micro electromechanical systems (MEMS) and especially in the development of atomic force microscopy. Their sensitivity and functional flexibility also make them an attractive technology for a new class of per- colative biosensors. Bio Nano Consulting has been working closely with Dr Rachel McKendry from the London Centre for Nanotechnology, University College London, who has dem- onstrated a novel and highly precise method to detect and quantify antibiotic– mucopeptide interactions.
Cantilever-based biosensing presents scien- tists with a range of advantages including removing the need for reporter ‘tags’ or external probes, and providing a one-step, high speed detection reaction that distinguishes between biomolecules. Cantilevers also offer the ability to create arrays of sensors, which can be used to screen multiple drug–target interactions along with reference coatings in parallel, under identical experimental conditions. There is also no limitation of the detection due to mass, as noted with other techniques. In summary, cantilever-based sensing provides a unique and highly precise method of measuring small-molecule drug-binding interactions and, are ideal for parallelisation enabling high-throughput screening of thousands of drugs per hour.
Benchmarking
Dr McKendry and colleagues have recently published a benchmarking study, in Nature Nanotechnology [1], into this novel application for micro-cantilever technology, showing that cantilever arrays have the sensitivity to detect and quantify the binding affinity of the antibiotic vancomycin to the drug– target mucopeptide analogues: Lysine-DAlanine-D-Alanine – found in vancomycin- sensitive bacteria, and Lysine-D-Alanine-D- Lac - found in vancomycin-resistant bacteria.
The studies clearly demonstrated that the system can detect the subtle binding changes associated with drug resistance (the deletion of a single hydrogen bond from the drug binding pocket in this in- stance). Importantly, the minimum detect- able vancomycin concentration was 10 nM and the technology can detect and quantify vancomycin directly in blood serum at clinically relevant concentrations. This is valu- able for pharmacokinetic / dynamic drug profiling, personalised medicine and forensic applications.
Cantilever bending
The cantilever system works by detecting the extent of bending in the arm, which is directly proportional to the number and strength of the binding events occurring along its length. The full detail of how the binding events lead to the necessary repulsive-compressive forces is the subject of much debate and interest [2-10]. The sen- sitivity of system is matched by the capability to differentiate between different antibiotics. As well as showing that cantilevers are a very useful tool in antibiotic research, the research conducted by Dr McKendry and co-workers has provided a new framework for understanding and eventually engineering the response of cantilevers to biochemical signals.
The Appliance of Science
Bio Nano Consulting has successfully applied this cantilever-array technology during commercial investigations into the mechanism of action of a novel antibiotic, proving that this microscale technology offers a quantitative method with nanomolar sensitivity that can be used to aid the characterisation of the interactions between lead compounds and their targets.
References:
1. Ndieyira, J. W. et al. Nanomechanical detection of antibiotic–mucopeptide binding in a model for superbug drug resistance. Nature Nanotech. 3, 691-696 (2008).
2. Fritz, J. et al. Translating biomolecular recognition into nanomechanics. Science 288, 316–318 (2000).
3. McKendry, R. A. et al. Multiple label-free biodetection and quantitative DNA-binding assays on a nanomechanical cantilever array. Proc. Natl Acad. Sci. USA 99, 9783–9788 (2002).
4. Savran, C. A., Knudsen, S. M., Ellington, A. D. & Manalis, S. R. Micromechanical detection of proteins using aptamer-based receptor molecules. Anal. Chem. 76, 3194–3198 (2004).
5. Calleja, M. et al. Highly sensitive polymer-based cantilever-sensors for DNA detection. Ultramicrosc. 105, 215–222 (2005).
6. Shu, W. et al. DNA molecular motor driven micromechanical cantilever arrays. J. Am. Chem. Soc. 127,