A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition
A Mass Spectrometry-Based Assay for Improved Quantitative Measurements of Efflux Pump Inhibition
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DOI：
10.1371/journal.pone.0124814
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发表时间：
2015-05-11
期刊：
影响因子：
3.7
通讯作者：
Cech, Nadja B.
中科院分区：
文献类型：
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作者：
Brown, Adam R.;Ettefagh, Keivan A.;Cech, Nadja B.
细菌外排泵是主动转运蛋白，负责对选定的杀菌剂和抗生素产生抗性。研究表明，在一些高致病性细菌中，包括耐甲氧西林金黄色葡萄球菌，外排泵的产生是上调的。因此，鉴定新的细菌外排泵抑制剂是一个非常有趣的话题。现有的评价外排泵抑制活性的方法依赖于外排泵底物的荧光。当使用这些实验来评估植物提取物和一些纯化化合物的外排泵抑制活性时，我们观察到严重的光干扰导致假阴性结果。为了解决这一问题，建立了一种新的基于质谱法的细菌外排泵抑制定量测量方法。本实验对加拿大水蛇粗提物的外排泵抑制活性进行了评价，并比较了几种纯类黄酮的外排泵抑制活性。槲皮素的IC50值为75 μ g/mL，荧光法显示槲皮素完全无活性。其他类黄酮（芹菜素、山奈酚、鼠李素、木犀草素、杨梅素）也有活性，IC50值在19 ~ 75 μ g/mL之间。本文所描述的测定方法可用于未来的筛选工作，以确定外排泵抑制剂，特别是在光学干扰排除依赖于荧光的方法的应用的情况下。
Bacterial efflux pumps are active transport proteins responsible for resistance to selected biocides and antibiotics. It has been shown that production of efflux pumps is up-regulated in a number of highly pathogenic bacteria, including methicillin resistant Staphylococcus aureus. Thus, the identification of new bacterial efflux pump inhibitors is a topic of great interest. Existing assays to evaluate efflux pump inhibitory activity rely on fluorescence by an efflux pump substrate. When employing these assays to evaluate efflux pump inhibitory activity of plant extracts and some purified compounds, we observed severe optical interference that gave rise to false negative results. To circumvent this problem, a new mass spectrometry-based method was developed for the quantitative measurement of bacterial efflux pump inhibition. The assay was employed to evaluate efflux pump inhibitory activity of a crude extract of the botanical Hydrastis Canadensis, and to compare the efflux pump inhibitory activity of several pure flavonoids. The flavonoid quercetin, which appeared to be completely inactive with a fluorescence-based method, showed an IC50 value of 75 mu g/mL with the new method. The other flavonoids evaluated (apigenin, kaempferol, rhamnetin, luteolin, myricetin), were also active, with IC50 values ranging from 19 mu g/mL to 75 mu g/mL. The assay described herein could be useful in future screening efforts to identify efflux pump inhibitors, particularly in situations where optical interference precludes the application of methods that rely on fluorescence.