A novel strategy to screen inhibitors of multiple aminoglycoside-modifying enzymes with ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry

摘要

Resistance to aminoglycoside antibiotics occurs primarily as a result of aminoglycoside-modification enzymes (AMEs) that modify the antibiotics. In this work, a novel strategy to combat the effects of antibiotic resistance was developed by screening multiple AMEs inhibitors with ultra-high performance liquid chromatography-quadrupole-time-of-flight mass spectrometry (UHPLC-QTOF MS). The method screened inhibitors of three AMEs (AAC(6')-APH(2 ''), AAC(6') and APH(2 '')) simultaneously through measuring the acetyltransferase activity and phosphotransferase activity of AAC(6')-APH(2 '') enzyme in a single assay. Screening inhibitors of multiple targets could greatly improve the screening efficiency at early-stages of drug discovery. In this study, enzyme reaction conditions including cosubstrate, enzyme concentration and cosubstrate concentration were optimized. The inhibition constants (K-i) for two known inhibitors, paromomycin and quercetin, were determined to be 1.23 and 20.27 mu M, respectively. The assay was further validated through the determination of a high Z' factor value of 0.73. The developed assay was applied to screen a chemical library against bifunctional AAC(6')-APH(2 '') enzyme. Using this assay, two pyrimidinyl indole derivatives were found to be potent, and effective AAC(6')-APH(2 '') inhibitors. The assay of exploring the selective inhibitory effect on two AAC(6')-APH(2 '') active sites was further performed. Two pyrimidinyl indole derivatives were found to exhibit striking inhibitory activities on AAC(6'). (C) 2018 Elsevier B.V. All rights reserved.