Early stage drug discovery screening for novel compounds active against the persister phenotype in Burkholderia thailandensis.

Abstract

Many pathogenic microorganisms are believed to stochastically switch into low metabolic states that display resistance to supra-lethal levels of antibiotics. These so-called “persister” cells have been associated with recurrent infections and the development of antibiotic resistance. Whilst a compound that eliminates Staphylococcus aureus persister cells has been described, it is not active against Gram-negative bacteria. The aim of my PhD project was to develop a high-throughput assay for compounds that eradicate persister cells in the -proteobacterium Burkholderia thailandensis. Further to this, I aimed to develop “hit” compounds from screening into lead series through investigation of structure activity relationships and, use a chemical genetics approach to elucidate potential mechanisms of action. I developed a phenotypic assay to identify compounds that eradicate persister cells. The assay was based on the reduction of the resazurin based dye PrestoBlue. Optimization of the assay gave a Z’ prime of 0.41 when screened in high throughput at the DDU. Screening of the library of 61,250 compounds identified 2,127 compounds that gave a statistically significant reduction in persister cell numbers. Follow-up assays highlighted 29 compounds with a pIC50 greater than five. Detailed investigation allowed me to down select to six “best in class” compounds, which included the licensed drug chloroxine. A time dependent killing assay showed that chloroxine reduced levels of persister cells by three orders of magnitude over 72 hours (P = 0.01). Hit expansion around chloroxine using commercially available compounds did not identify any more potent compounds, but did highlight key features of the molecule for activity. Assay protocols were provided to collaborators at DSTL who were able to iv show that chloroxine is also active against persister cells formed by the tropical pathogen and Tier 1 biological agent Burkholderia pseudomallei. Investigations into the mechanism of action of chloroxine used Next Generation Sequencing of an over expression library, identifying two putative genes involved in inhibition of persister cells by chloroxine. My findings demonstrate a phenotypic assay against persister cells in Gram-negative bacteria, which has the power to identify potent anti-persister agents to assist in chemotherapy. Structural activity relationship and mechanism of action investigations have indicated lead series and genetic starting points for future development of this research. My PhD project has concluded with sufficient data for continuation of research following a number of leads and is at an ideal stage for instigation of a medicinal chemistry program for development of chloroxine as a clinical option for treatment of persistent melioidosis.