A fluorescein-labeled AmpC β-lactamase allows rapid characterization of β-lactamase inhibitors by real-time fluorescence monitoring of the β-lactamase-inhibitor interactions.

Rapid emergence of class C β-lactamases has urged an immediate need for developing class C β-lactamase specific inhibitors for effective clinical treatment. To facilitate the development of effective class C β-lactamase inhibitors, we propose a new approach for a rapid analysis of the interaction of AmpC β-lactamase and its inhibitors using our recently developed V211Cf fluorescent β-lactamase biosensor during drug screening. Since the fluorescein of V211Cf can report the local environment change in the active site of AmpC β-lactamase, fluorescence responses of V211Cf toward its substrates/inhibitors can provide real-time traces of the dynamic change of the interaction of the β-lactamase with its substrates/inhibitors. In this study, we found that V211Cf displayed distinct fluorescence signal patterns toward different kinds of inhibitors (including clavulanic acid, sulbactam, tazobactam and 2-thiopheneboronic acid) due to the differences in their interactions with β-lactamase. V211Cf not only enables a high throughput screening for inhibitors but can also provide a rapid preliminary indication on the inhibitor's potency and stability to β-lactamase's hydrolytic action as well as how the inhibitors interact with the target enzyme, thereby speeding up the drug discovery and development cycle of class C β-lactamase inhibitors.