5-Carboxytetramethylrhodamine-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 analogue 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 analogue of ampicillin, 5-carboxytetramethylrhodamine-ampicillin (5-TAMRA-ampicillin), was useful as the indicator in a continuous fluorescence anisotropy-based kinetic assay for inhibition of a soluble construct of PBP2 from E. coli. The assay enables measurement of the bimolecular rate constant for inhibition kinact/Ki. This measurement was made for representative drugs from four classes of β-lactams and for the diazabicyclooctenone ETX2514. 5-TAMRA-ampicillin was also useful in a fluorescence anisotropy-based assay for P. aeruginosa PBP2 and in fluorescence intensity-based assays with PBP1a and PBP3 from P. aeruginosa and A. baumannii and PBP3 from E. coli.