title: A fluorescein-labeled AmpC β-lactamase allows rapid characterization of β-lactamase inhibitors by real-time fluorescence monitoring of the β-lactamase-inhibitor interactions.
abstract: 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.
authors.affiliation: Department of Applied Biology and Chemical Technology and the State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
authors.forename: Man-Wah
authors.initials: MW
authors.lastname: Tsang
authors.affiliation: Department of Applied Biology and Chemical Technology and the State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
authors.forename: Pak-Ho
authors.initials: PH
authors.lastname: Chan
authors.affiliation: Department of Applied Biology and Chemical Technology and the State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
authors.forename: Sze-Yan
authors.initials: SY
authors.lastname: Liu
authors.affiliation: Department of Applied Biology and Chemical Technology and the State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China. kwok-yin.wong@polyu.edu.hk.
authors.forename: Kwok-Yin
authors.initials: KY
authors.lastname: Wong
authors.affiliation: Department of Applied Biology and Chemical Technology and the State Key Laboratory of Chirosciences, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China. thomas.yun-chung.leung@polyu.edu.hk.
authors.forename: Yun-Chung
authors.initials: YC
authors.lastname: Leung
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D015374:Biosensing Techniques / Q000379:methods*; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004795:Enzyme Stability; D005452:Fluoresceins / Q000737:chemistry*; D057166:High-Throughput Screening Assays; D013194:Staining and Labeling / Q000379:methods; D065093:beta-Lactamase Inhibitors / Q000494:pharmacology*; D001618:beta-Lactamases / Q000737:chemistry* / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005452:Fluoresceins; D065093:beta-Lactamase Inhibitors; C048795:AmpC beta-lactamases; D001618:beta-Lactamases
keywords: Class C β-lactamase; Fluorescein; Fluorescent biosensor; Pharmaceutical industry; β-lactamase inhibitor