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

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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 (Ki) for two known inhibitors, paromomycin and quercetin, were determined to be 1.23 and 20.27 μM, 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’).