Bacterial defiance as a form of prodrug failure

Brandon Alexander Holt; Gabriel A. Kwong

To measure drug cytotoxicity, we quantified the percent of viable bacteria when exposed to unlocked AMP drug doses ranging from nanomolar to millimolar concentrations and observed a 5 spike in cytotoxicity in the micromolar range (Fig. S1A) . To calculate bacterial growth rate constants, we incubated bacteria in broth or water at 37 o C and measured OD600 over the course of 8 hours, revealing a higher growth rate in broth consistent with published values 35, 56 (3.0 > 0.1 h -1 ) (Fig. S1B) . To quantify enzymatic velocity, we measured proteolytic velocity (moles L -1 s -1 ) under substrate concentrations ranging from 0-200 uM and fit the Michaelis-Menten equations 10 to this data to calculate kcat and Km (Fig. S1C ). Using these measured parameters (Table S2) , we generated kinetic curves for bacteria number and drug concentration over time. The model showed that after a period of initial bacterial growth, the lagging drug population eliminated the infection burden. While the initial reservoir of locked drug concentration was ~200 µM, the unlocked drug never rose above 5 µM (Fig. S1D) , revealing that this system maintains the minimal 15 drug concentration required to kill the infection. The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/556951 doi: bioRxiv preprint The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/556951 doi: bioRxiv preprint