A general method for determining bacterial uptake of compounds independent of antibacterial activity would be a valuable tool in antibacterial drug discovery. LC-MS/MS assays have been described, but it has not been shown whether the data can be used directly to inform medicinal chemistry. We describe the evaluation of an LC-MS/MS assay measuring association of compounds with bacteria, using a set of over a hundred compounds (inhibitors of NAD-dependent DNA ligase, LigA) for which in vitro potency and antibacterial activity had been determined. All compounds were active against an efflux-deficient strain of Escherichia coli with reduced LigA activity (E. coli ligA251 ΔtolC). Testing a single compound concentration and incubation time, we found that, for equipotent compounds, LC-MS/MS values were not predictive of antibacterial activity. This indicates that measured bacteria-associated compound was not necessarily exposed to the target enzyme. Our data suggest that, while exclusion from bacteria is a major reason for poor antibacterial activity of potent compounds, the distribution of compound within the bacterial cell may also be a problem. The relative importance of these factors is likely to vary from one chemical series to another. Our observations provide directions for further study of this difficult issue.

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评估LC-MS / MS以测量细菌内化合物的积累

确定与抗菌活性无关的化合物对细菌的摄取的一般方法将是发现抗菌药物的有价值的工具。已经描述了LC-MS / MS测定法，但是还没有显示数据是否可以直接用于药物化学研究。我们描述了一种LC-MS / MS分析方法的评估，该方法使用一组一百多种已经确定了体外效价和抗菌活性的化合物（NAD依赖性DNA连接酶抑制剂，LigA抑制剂）来评估化合物与细菌的缔合。所有化合物有效对抗的外排缺陷株大肠杆菌具有降低的LIGA活性（大肠杆菌ligA251 Δ TOLC）。通过测试单个化合物的浓度和孵育时间，我们发现，对于等价化合物，LC-MS / MS值不能预测抗菌活性。这表明所测量的细菌相关化合物不一定暴露于目标酶。我们的数据表明，尽管排除细菌是有效化合物抗菌活性差的主要原因，但化合物在细菌细胞内的分布也可能是一个问题。这些因素的相对重要性可能会因一个化学系列而异。我们的观察为进一步研究这个难题提供了指导。