Screening for the inhibitor of carbapenem hydrolyzing class D beta-lactamase

• Main Authors: Bo-Li Wang, 王博立
• Other Authors: Te-Li Chen
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/51641680951672842491

碩士 === 國立陽明大學 === 臨床醫學研究所 === 101 === 　　Acinetobacter baumannii is notorious for its carbapenem resistance, and limited therapeutic options are available for patients who are infected with this organism. The overproduction of OXA-51-like β-lactamases, a subgroup of carbapenem hydrolyzing class D β-lactamase, is one of the major mechanisms of carbapenem resistance in A. baumannii, especially in Taiwan. Development of the OXA-51-like β-lactamase inhibitors could help in the treatment of patients infected by carbapenem resistant A. baumannii. This study aims to optimize the OXA-51-like β-lactamase purification system and to develop a targeted-based assay for screening of chemicals library to discover potential OXA-51-like β-lactamase inhibitors. First, we purified OXA-51-like β-lactamase using the His-tagged and GST-tagged protein purification system. We found a higher yield, better purity, and stronger capability of nitrocefin hydrolysis using the GST-tag purification system. The optimized condition was: induction of the bacterial (growth until OD600=0.8) using 160μM IPTG at 23°C for 17hr. Then we screened 324 extracts of Chinese herb and 1280 compounds of Sigma LOPAC library. We found that two extracts of Chinese herb and 12 compounds of Sigma LOPAC library could inhibit nitrocefin hydrolysis by GST-tagged OXA-51-like β-lactamase. Among them, only BLI-489 could inhibit imipenem hydrolysis by not only OXA-51-like but also OXA-23, 24, 58 β-lactamases in an A. baumannii cell-based system. The results showed that platform could discover the potential inhibitor of carbapenem hydrolyzing class D β-lactamase.