5-Carboxytetramethylrhodamine-Ampicillin (5-TAMRA-ampicillin) Fluorescence Anisotropy-based Assay of Escherichia coli Penicillin-Binding Protein 2 Transpeptidase Inhibition

The high-molecular mass penicillin-binding proteins (PBPs) are the essential targets of the β-lactam classes of antibacterial drugs. In the Gram-negative pathogen Escherichia coli, these include PBP1a, PBP1b, PBP2, and PBP3. Techniques that enable facile measurement of the potency of inhibition of these targets are valuable for understanding structure-activity relationships in programs aimed at discovering new antibiotics to combat drug-resistant infections. Continuous, fluorescence anisotropy-based assays for inhibition of soluble constructs of PBP1a, PBP2 and PBP3 from the serious Gram-negative bacterial pathogens Pseudomonas aeruginosa and Acinetobacter baumannii, and PBP3 from E. coli using the fluorescent phenoxypenicillin analog BOCILLIN FL have been described previously, but this technique was not useful for PBP2 from E. coli due to a lack of change in fluorescence anisotropy or intensity upon reaction. Here we report that a fluorescent analog of ampicillin, 5 carboxytetramethylrhodamine-ampicillin...