Prakash Singh Maya
Chemical and Screening Sciences, Wyeth Research, 401 N. Middletown Road, Pearl River, NY 10965, USA.
J Microbiol Methods. 2006 Oct;67(1):125-30. doi: 10.1016/j.mimet.2006.03.011. Epub 2006 May 2.
In the search for antibacterial agents with a novel mode-of-action (MOA) many targeted cellular and cell-free assays are developed and used to screen chemical and natural product libraries. Frequently, hits identified by the primary screens include compounds with nonspecific activities that can affect the integrity and function of bacterial membrane. For a rapid dereplication of membrane-active compounds, a simple method was established using a commercially available Live/Dead(R) Bacterial Viability Kit. This method utilized two fluorescent nucleic acid stains, SYTO9 (stains all cells green) and propidium iodide (stains cells with damaged membrane red) for the drug-treated bacterial cells. The cells were then either examined visually by fluorescence microscopy or their fluorescence emissions were recorded using a multi-label plate reader set to measure emissions at two different wavelengths. The ratio of green versus red was compared to a standard curve indicating the percentage of live versus dead bacteria. Nine known antibiotics and 14 lead compounds from various antibacterial screens were tested with results consistent with their MOA.
在寻找具有新型作用模式（MOA）的抗菌剂时，人们开发了许多靶向细胞和无细胞检测方法，并用于筛选化学和天然产物文库。通常，初次筛选确定的命中化合物包括具有非特异性活性的化合物，这些活性会影响细菌膜的完整性和功能。为了快速去除膜活性化合物的重复物，建立了一种使用市售的活/死细菌活力试剂盒的简单方法。该方法利用两种荧光核酸染料，SYTO9（将所有细胞染成绿色）和碘化丙啶（将膜受损的细胞染成红色）对药物处理的细菌细胞进行染色。然后通过荧光显微镜目视检查细胞，或者使用设置为在两个不同波长下测量发射的多标记微孔板读数器记录它们的荧光发射。将绿色与红色的比率与指示活细菌与死细菌百分比的标准曲线进行比较。对9种已知抗生素和来自各种抗菌筛选的14种先导化合物进行了测试，结果与它们的作用模式一致。
