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. The authors report the use of a recently developed V211Cf fluorescent β‐lactamase biosensor for real‐time fluorescence analysis of the interaction of β‐lactamase inhibitors and their β‐lactamase target during the drug screening process. They show that V211Cf not only gives rise to different pattern of fluorescence signals according to the interaction between the inhibitor and the enzyme, but also allows drug screening and initial analysis for the interaction between the inhibitor and β‐lactamase drug target to be performed at the same time, thereby facilitating the development of the class C β‐lactamase inhibitors.