Bacterial defiance as a form of prodrug failure

Brandon Alexander Holt; Gabriel A. Kwong

The rising threat of antibiotic failure has motivated the classification of resistance mechanisms to improve the design of future treatment strategies. In this work, we identified a prodrug-specific subclass of resistance, which we named defiance. To study bacterial defiance, we synthesized an AMP prodrug that was activated by bacterial protease activity and successfully 10 killed E. coli. However, we found that this prodrug failed in conditions where the bacteria grew faster than the rate at which they activated the drug. We developed a mathematical model of the bacteria-prodrug competition and derived a dimensionless constant (BAH), which we used to perfectly predict the onset of the defiance phenotype. These studies revealed that both environmental (e.g., temperature, available nutrients) and pharmacokinetic (e.g., activation rate 15 kcat) parameters can be tuned to engineer successful prodrug therapies. In future work, this information could be leveraged to improve the efficacy of existing prodrugs by tuning the prodrug decay constant (i.e., biological half-life), which directly changes the BAHcrit value. Alternatively, the catalytic efficiency of the prodrug substrate could be tuned to change the BAH value and increase the probability of success, which has been previously demonstrated by engineering 20 prodrug substrates with higher affinity for the enzymatic target 44, 45 .