Screening for broad spectrum antimicrobials with unknown targets

Abstract

The misuse of antibiotics combined with pathogen tolerance has led to an increase in patients with multidrug resistant infections, relapse of infections, and fewer effective treatments. We propose that prodrugs hold potential as the next broad spectrum antimicrobial. Prodrugs are able to diffuse into the cell where they are converted into a reactive compound by bacterial-specific enzymes. We developed a unique high-throughput screening assay to identify prodrugs. We hypothesized that since prodrugs have multiple targets they would rapidly abolish metabolism resulting in cell death. The viability dye alamar blue was used to test this hypothesis and measured the metabolism of cells challenged with antiseptics, antibiotics, and the prodrug Nitazol. We employed a high-throughput screen for broad spectrum antimicrobials using Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus), and candidate prodrug hits were tested for cytotoxicity and minimum inhibitory concentrations (MIC) against a panel of pathogens. A validation step confirmed a strain lacking a converting enzyme was more resistant to a prodrug and a strain over expressing a converting enzyme was more susceptible. Prodrugs appear to rapidly abolish metabolism and produce a distinct kinetic curve in the alamar blue reduction screen. Three compounds were identified as potential broad spectrum prodrugs from this screen. Compound ADC111, a nitrofuran compound, shows broad spectrum activity and mimimal cytotoxicity against four mammalian cell lines. In E. coli, ADC111 is converted by nitroreductases NfsA and NfsB. The second prodrug candidate, ADC112, is an 8-hydroxyquinoline, a class of broad spectrum antimicrobials where the mechanism of action is unknown. The third prodrug candidate, ADC113, does not belong to a known class of approved antimicrobials. Different classes of antimicrobials have different effects on bacterial metabolism, which can be differentiated using the alamar blue reduction screen. This screen can identify compounds with unknown targets.