Bacterial defiance as a form of prodrug failure

Brandon Alexander Holt; Gabriel A. Kwong

Antibiotic success is markedly improved by proper titration of drugs, as overdosing leads to off-target toxicity and underdosing increases the likelihood of pathogens developing resistance 7 . However, optimal drug doses are difficult to achieve over the course of treatment because infection burden changes dynamically over time, creating a moving target 7, 8 . Prodrugs, which represent ~10% of all FDA-approved drugs in the last decade, are a promising solution 20 because they may be automatically titrated by a disease-related activation mechanism, increase bioavailability, and reduce the risk of off-target effects 9 . For example, prodrugs such as ganciclovir 10 and isoniazid 11 are administered as biologically inactive forms and are enzymatically activated by the pathogen. Here, we use prototypic auto-titrating prodrugs comprising cationic antimicrobial peptides (AMP), which evolved as a part of innate immunity and display broad-25 spectrum and prokaryote-specific antimicrobial activity 12 . Cationic AMPs act by disrupting bacterial membranes and inducing inflammatory responses 13 , but suffer from off-target toxicity and low stability in vivo 14, 15 . Our AMP prodrugs are activated directly by bacterial proteases, which are a major cause of virulence [16] [17] [18] [19] [20] [21] during infections and important targets for antibiotic development 22 . Of these proteases, outer membrane protease T (OmpT) is a membrane-bound 5 bacterial protein that is widely conserved across gram-negative bacteria of the Enterobacteriaceae family and recognizes a variety of targets that contribute to its virulence (e.g., plasminogen) 18, 19, [23] [24] [25] . In this work, we design cationic AMPs that are locked by charge complexation with anionic peptides and connected with a protease-cleavable linker substrate [26] [27] [28] that is recognized by E. coli protease OmpT, such that increasing concentrations of bacteria 10 activate higher concentrations of free drug.