Small Molecule Hits, the Big Screen: Identification of Novel Inhibitors of Nitric Oxide Defense in E. coli

Abstract

Anti-virulence therapies are treatments used to stop infections rather than kill the pathogen itself. To increase the knowledge base of possible anti-virulence therapies against the bacteria Escherichia coli, the nitric oxide detoxification network was studied by designing a high throughput screen to find small molecules that could disrupt this immune evasion response of E. coli. If E. coli cannot detoxify or clear nitric oxide (a molecule used by phagosomes in the immune response to irreversibly damage pathogens), it cannot grow, and this fact was used to design a high throughput screen. A statistically robust high throughput screening protocol was developed to find inhibitors that could only prevent growth when working synergistically with nitric oxide. This study showed that by implementing a rigid threshold for consideration of hits, very few false positives clouded the final results. The readily available small molecules of 2-mercaptobenzothiazole and biochanin A were validated as nitric oxide defense network disruptors by measuring the clearance profiles of E. coli upon treatment with these small molecules. The families of these compounds were investigated to begin a structure-activity relationship study. Other molecules that were not readily available by chemical vendors have also popped out as hits in this study, and future studies will validate them as hits or false positives using the same protocol in this study.