Development and characterisation of vectors for delivery of antibiotic transcription factor decoys (tfd's) to Gram negative bacteria

A range of novel transcription factor decoy (TFD) based antibiotics have been developed that target bacterial cells in a species specific manner. At most sites where infection occurs however, non-target species are present. The aim of this project is to study the impact of these species-specific antibiotics on both target and non-target bacterial cells. Combining TFDs with a peptide or mimetic delivery system, we will obtain models, supported by experimental data, for how these antibiotics interact with specific targets and how this in turn affects the dynamics of bacterial communities whose composition is a determinant of disease. These tools will aid the design of future therapeutics with greater efficacy and fewer side effects. TFDs are being developed by Procarta as a novel type of antibacterial agent. TFDs are DNA copies of key regulatory sites that competitively inhibit transcription factor binding and so control bacterial gene expression. Procarta has demonstrated that they block the expression of essential genes at nanomolar concentrations and so kill bacteria. Using biophysical methods, Dr. Mason (AJM) has described properties of cationic alpha-helical AMPs that promote specific interaction with bacterial membranes and enable the peptides to bind, penetrate, disorder and cross these barriers. Ongoing research has produced peptides that are effective against Gram negative bacteria including E. coli and P. aeruginosa at low micromolar to high nanomolar concentrations. TFDs targeted to alternative sigma transcription factors prevent induction of the bacterial stress response and act as potent antibacterials in vitro and in vivo. Delivery is mediated by antimicrobial peptides which preferentially permeabilise bacterial membranes and can carry cargoes into a broad range of bacteria. Procarta has identified a short peptide capable of efficiently transfecting TFDs into S. aureus and Escherichia coli. The present proposal will develop novel peptide vectors to deliver TFDs targeting Gram-negative pathogens. This will involve using expertise available at King's (AJM and Dr Sukhi Bansal) to develop an improved formulation for the TFDs where the peptide component is replaced with a peptidomimetic (offering greater stability, reduced toxicity and cost) and using this molecule in animal models to create a pre-clinical data package validating the use of TFDs to treat as a relevant model Extended-Spectrum beta-lactamase E. coli infections. At King's, AJM is creating a multidisciplinary biophysical platform which aims to generate computer models, supported by experimental data, capable of predicting the action of antibiotic peptides and vectors against target cell and internal membranes. The TFD/peptide system will be included in this program in order that more effective and specific complexes can be produced. AJM and Dr Ken Bruce share an interest in understanding and combating bacterial infections in the cystic fibrosis lung and therefore, following on from the development of peptide vectors, the project will then involve the further development of an existing biofilm model for P. aeruginosa and mixed bacterial communities against which the formulated TFDs can be tested. The effect of specific antibacterial action on the biofilm and its constituents will be determined experimentally so that an in silico model can be generated. This model will describe the bacterial population dynamics within mixed communities enabling the response to the known action of antibiotics with varying spectra of action to be predicted. Such information will enable a new era of antibiotic therapy where the bacterial community associated with each individual patient is considered allowing more appropriate therapeutic regimes to be selected.