Microtechnology Focus
Antibiotic Resistance - Nanotech to the Rescue: Nanomechanical detection of drug-target interactions
Drug resistance has evolved from being an infrequent and manageable occurrence in the treatment of microbial infections, to a healthcare problem on a global scale. The last five years has seen a meteoric rise in the cases of life-threatening infections by mutated forms of common bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus (VRE). Eradication of such multi-drug resistant (MDR) strains is proving difficult since there are not enough effective antimicrobials on the market, and very few in the development pipelines. There is though, a concerted effort to develop new compounds capable of combating these infections effectively. A novel drug-target binding detection system is proving itself to be a big hit in this search. It is based on micro-cantilever technology and provides exquisite information on the nature of a drug-target interaction. Furthermore, it can be applied on a high throughput basis making the search for new products much quicker too.
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The differential measurements generated enabled clear discrimination between the two peptide sequences. This showed that the system can detect the subtle binding changes induced by the deletion of a single hydrogen bond
Author Details:
Dr Mike Fisher/Dr David Sarphie Bio Nano Consulting
www.bio-nano-consulting.com Tel: +44 (0)207 268 3032
Email:
admin@bio-nano-consulting.com Figure 1: Binding of Vancomycin to mucopeptide receptors, showing the single atom change that causes Van resistance. BASIC PRINCIPLES
By immobilising a ligand on one side of a cantilever and adding the relevant receptor in solution, the cantilever bends in response to the change in surface stress when the ligand and receptor interact. This nanomechanical biosensing transduction mechanism has been used for measuring a number of biochemically specific interactions such as sequence-specific DNA hybridisation, single base mismatches, DNA quadruplex, protein recognition and has also been used for the detection of interferon-alpha- induced I-8U gene expression in total human RNA - a potential marker for melanoma progression and viral infections. This technology has now been applied very successfully to quantify drug–target binding interactions.
Cantilever advantages
Cantilever-based sensing offers a number of advantages over existing technologies. For example it does not require the use of any reporter ‘tags’ or external probes, and can therefore be used to detect biomolecules rapidly in a single-step reaction. Arrays of cantilever sensors can be constructed to screen multiple drug–target interactions and reference coatings in parallel, under identical experimental conditions. Furthermore, quantitative ligand–receptor binding constants can be measured on cantilever arrays. Due to the nature of the detection, the nanomechanical signal generated from a cantilever is not limited by mass, a restriction commonly associated with evanescent techniques such as surface plasmon resonance.
In summary, cantilever-based sensing provides a unique and highly precise method of measuring small-molecule drug-binding interactions and is ideal for parallelisation, enabling high-throughput screening of thousands of drugs per hour.
EXAMPLE APPLICATION
Vancomycin (Van) is a vital therapeutic drug used worldwide for the treatment of infections with Gram- positive bacteria, particularly those Staphylococci and Enterococci responsible for post-surgical infections.
It is also, essentially, the last chance antibiotic in the battle against MRSA super-bug infections. Van is a very potent bactericidal compound, binding to the C-terminus of peptidoglycan mucopeptide precursors terminating in the sequence ‘Lysine—D-Alanine—D-Alanine’, as shown in Figure 1. As a result it blocks bacterial transpeptidases and transglycosylases, which catalyse the crosslinking of the growing bacterial cell wall, resulting in cell lysis. With the rapid turnover and mutation capabilities of many bacterial species, coupled with the misuse of antibiotics, resistance to Van has rapidly increased and continues to do so at an alarming rate. As a result, infections from Van resistant species now pose a major international public health problem, with growing morbidity and mortality. Developing resistance to Van in Enterococci only requires the change of an amide linkage to an ester linkage in the growing bacterial cell wall – a subtle change. This results in the deletion of a single hydrogen bond to the binding pocket, rendering the antibiotic therapeutically ineffective (Figure 1).
A recent series of investigations [1] by an international team of scientists led by Dr Rachel McKendry, London Centre for Nanotechnology, University College London, has demonstrated a novel and highly precise method to detect and quantify the binding of antibiotic–mucopeptide interactions.
INVESTIGATION SET-UP
Multiple arrays of eight rectangular silicon cantilevers were developed to probe the nanomechanics of antibiotic drug–target interactions. The cantilever arms were coated on one side with a thin film of gold and functionalised with alkanethiol self-assembled monolayers (SAMs) of either:
1. Lysine-D-Alanine-D-Alanine – found in Van-sensitive bacteria (DAla); or 2. Lysine-D-Alanine-D-Lac - found in Van-resistant bacteria - (DLac); or
3. Polyethylene glycol - PEG (control cantilever). The absolute deflection at the free end of each cantilever
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