Engineering steroid drugs to target bacterial respiration

1.1 - Context: Antibiotic resistance in bacterial pathogens is an escalating global problem and new strategies are urgently required to combat infections. We have very recently demonstrated that steroid drugs inhibit cytochrome bd, an emerging drug target that is found in the respiratory chains of a variety of bacterial pathogens. Furthermore, this study revealed that quinestrol, a prodrug used for hormone replacement therapy, is bacteriostatic towards multidrug-resistant E. coli and is bactericidal towards methicillin-resistant Staphylococcus aureus (MRSA). However, important gaps in our knowledge exist, such as the mode of inhibition, the cellular impact, the scope for improving specificity, and the potential for encapsulation approaches to deliver quinestrol and it's derivatives to the site of infection. This project seeks to extend our understanding of these key areas. 1.2 - Challenge addressed by project: The emergence of antimicrobial resistance in bacterial pathogens is making it increasingly difficult to treat infections, which is compounded by an inadequate supply of new antibiotic classes under development. This project will identify drugs that can be used for the treatment of bacterial infections where conventional antibiotics no longer work. The overarching aim of this project is to robustly assess the potential of steroid drugs to bind to cytochrome bd, a promising drug target that is crucial during infection for a broad range of bacterial pathogens. An overview of how this will be achieved is outlined in the aims and objectives below. 1.3 - Aims and objectives: Aim i): Characterise the mode of inhibition for quinestrol. Objective: Perform inhibition kinetics analyses to test the hypothesis that quinestrol is a competitive inhibitor of cytochrome bd with respect to quinol substrate. Aim ii): Gain mechanistic insights into the cellular impact of quinestrol. Objective: Expose E. coli and MRSA to various concentrations of quinestrol and assess the impact via measuring intracellular environmental changes using a range of molecular approaches. Sub-cellular tracking and quantitation of quinestrol will also be performed. Aim iii): Develop a series of quinestrol derivatives to improve specificity towards cytochrome bd complexes. Objective: Undertake structural modelling and in silico docking to design quinestrol derivatives, use click chemistry for synthesis, and enzyme kinetics to assess drug efficacy. Aim iv): Advance our structural understanding of how steroid inhibitors bind to cytochrome bd. Objective: Undertake structural investigations of protein ligand complexes using cryo-electron microscopy approaches. Aim v): Develop methods to deliver steroid drugs more rapidly to immune cells. Objective: Novel nanoencapsulation techniques will be developed to optimise steroid drug loading and to improve access to bacterial pathogens that have been internalised by immune cells.