5-Carboxytetramethylrhodamine-Ampicillin Fluorescence Anisotropy-Based Assay of Escherichia coli Penicillin-Binding Protein 2 Transpeptidase Inhibition

Abstract

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, 5carboxytetramethylrhodamine-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 k inact /K i . This measurement was made for representative drugs from four classes of βlactams 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.

Discovery of an Orally Available Diazabicyclooctane Inhibitor (ETX0282) of Class A, C, and D Serine β-Lactamases

“…Inhibition of E. coli PBP2 was measured according to the method published in Shapiro et al . A set of 2-fold serial dilutions of each inhibitor from 61.44 to 0.06 and 0 μM were prepared from a fresh solution of the compound dissolved in assay buffer consisting of 0.1 M sodium phosphate (pH 6.0) and 0.01% Triton X-100.…”

“…The second-order inactivation rate constant k inact /K i was calculated from the set of progress curves using Global Kinetic Explorer (Kintek) as previously described. 13 Inhibition of E. coli PBP2 was measured according to the method published in Shapiro et al 38 A set of 2-fold serial dilutions of each inhibitor from 61.44 to 0.06 and 0 μM were prepared from a fresh solution of the compound dissolved in assay buffer consisting of 0.1 M sodium phosphate (pH 6.0) and 0.01% Triton X-100. Inhibitor solution (2 μL) and 2 μL of 90 nM 5-TAMRA-ampicillin were added to the wells of a low-volume, shallow-well, black polystyrene, 384-well microplate (Corning).…”

Interactions of hydrolyzed β-lactams with the L1 metallo-β-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products

“…Kinetic parameters were calculated and analyzed using the Michaelis-Menten curve in GraphPad Prism 6 (GraphPad Software, La Jolla, CA, USA; www.graphpad.com ). The following wavelengths and extinction coefficients were used: tebipenem (Δε 297 = −9550 M −1 cm −1 ( 83 )); panipenem (Δε 297 = −7400 M −1 cm −1 ( 84 )); doripenem (Δε 296 = −7540 M −1 cm −1 ( 85 )); mecillinam (Δε 240 = −1100 M −1 cm −1 ( 86 )); imipenem (Δε 300 = −9000 M −1 cm −1 ( 87 )); meropenem (Δε 293 = −7600 M −1 cm −1 ( 87 )); biapenem (Δε 293 = −8630 M −1 cm −1 ( 87 )).…”

Live-Cell Profiling of Penicillin-Binding Protein Inhibitors in Escherichia coli MG1655

“…In this study, we report an optimized gel-based assay for the evaluation of 12 inhibitors in live, non-hypersusceptible E. coli MG1655. Historically, such assays have been carried out in vitro , using either pure proteins or cell lysates. ,− Results from these studies do not always translate to whole cells given the potential loss of native protein–protein interactions, as well as possible protein unfolding (i.e., the selectivity profile of a given inhibitor differs between live cells and lysates). Indeed, it has been demonstrated that Bocillin-FL labeling profiles in lysates are often missing some of the PBPs.…”