Department of Applied Biology and Chemical Technology, Central Laboratory of the Institute of Molecular Technology for Drug Discovery and Synthesis, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China.
Anal Chem. 2011 Mar 15;83(6):1996-2004. doi: 10.1021/ac102595r. Epub 2011 Feb 21.
Class C β-lactamases mediate antibiotic resistance in bacteria by efficiently hydrolyzing a broad range of β-lactam antibiotics. With their clinical significance and the lack of commercially available effective inhibitors, development of class C β-lactamase inhibitors has become one of the recent hot issues in the pharmaceutical industry. In this paper, we report the protein engineering of a fluorescent Amp C β-lactamase mutant designated as V211Cf for the in vitro screening of class C β-lactamase inhibitors. When a fluorescein (f) was incorporated at the entrance of the enzyme's active site (position 211), Amp C β-lactamase from Enterobacter cloacae P99 was tailor-made into a novel fluorescent biosensing protein that could display a fluorescence enhancement upon binding with its β-lactam substrates/inhibitors. With its catalytic activity close to the wild-type level, V211Cf can act as a "natural" fluorescent drug target for screening small binding molecules. In addition, V211Cf can allow specific detection for its active-site binding molecules and discriminate them from nondruglike molecules in the screen. Furthermore, V211Cf is amenable to a high throughput format. Taken together, V211Cf demonstrates the potential as an efficient tool for screening class C β-lactamase inhibitors and facilitates the discovery of therapeutics that can combat the clinically important class C β-lactamases.
C 类 β-内酰胺酶通过高效水解广泛的 β-内酰胺抗生素来介导细菌的抗生素耐药性。由于其临床意义以及缺乏商业上可获得的有效抑制剂，因此开发 C 类 β-内酰胺酶抑制剂已成为制药行业的近期热点问题之一。在本文中，我们报告了荧光 Amp C β-内酰胺酶突变体 V211Cf 的蛋白质工程，该突变体用于体外筛选 C 类 β-内酰胺酶抑制剂。当在酶活性位点的入口处（位置 211）掺入荧光素（f）时，来自阴沟肠杆菌 P99 的 Amp C β-内酰胺酶被定制成一种新型荧光生物传感蛋白，当与β-内酰胺底物/抑制剂结合时，该蛋白可以显示出荧光增强。V211Cf 的催化活性接近野生型水平，因此可以作为筛选小分子结合物的“天然”荧光药物靶标。此外，V211Cf 可以特异性检测其活性位点结合分子，并将其与筛选中的非药物样分子区分开。此外，V211Cf 适用于高通量格式。总之，V211Cf 有望成为筛选 C 类 β-内酰胺酶抑制剂的有效工具，并有助于发现可以对抗临床上重要的 C 类 β-内酰胺酶的治疗方法。
