k_inact / K_I Value Determination for Penicillin-Binding Proteins in Live Cells

Penicillin-binding proteins (PBPs) are an essential family of bacterial enzymes that are inhibited by the β-lactam class of antibiotics. PBP inhibition disrupts cell wall biosynthesis, which results in deficient growth and proliferation, and ultimately leads to lysis. IC ₅₀ values are often employed as descriptors of enzyme inhibition and inhibitor selectivity but can be misleading in the study of time-dependent, irreversible inhibitors. Due to this disconnect, the second order rate constant k _inact / K _I is a more appropriate metric of covalent inhibitor potency. Despite being the gold standard measurement of potency, k _inact / K _I values are typically obtained from in vitro assays, which limits assay throughput if investigating an enzyme family with multiple homologs (such as the PBPs). Therefore, we developed a whole-cell k _inact / K _I assay to define inhibitor potency for the PBPs in Streptococcus pneumoniae using the fluorescent activity-based probe Bocillin-FL. Our results align with in vitro k _inact / K _I data and show a comparable relationship to previously established IC ₅₀ values. These results support the validity of our in vivo k _inact / K _I method as a means of obtaining a full picture of β-lactam potency for a suite of PBPs.