von Gundlach A R, Garamus V M, Gorniak T, Davies H A, Reischl M, Mikut R, Hilpert K, Rosenhahn A
Analytical Chemistry - Biointerfaces, Ruhr-University Bochum, NC4, Universitätsstr, 150, 44780 Bochum, Germany.
Helmholtz-Zentrum Geesthacht, Zentrum für Material- und Küstenforschung GmbH, Max-Planck-Straße 1, 21502 Geesthacht, Germany.
Biochim Biophys Acta. 2016 May;1858(5):918-25. doi: 10.1016/j.bbamem.2015.12.022. Epub 2015 Dec 28.
Multi-drug resistant bacteria are currently undermining our health care system worldwide. While novel antimicrobial drugs, such as antimicrobial peptides, are urgently needed, identification of new modes of action is money and time consuming, and in addition current approaches are not available in a high throughput manner. Here we explore how small angle X-ray scattering (SAXS) as high throughput method can contribute to classify the mode of action for novel antimicrobials and therefore supports fast decision making in drug development. Using data bases for natural occurring antimicrobial peptides or predicting novel artificial peptides, many candidates can be discovered that will kill a selected target bacterium. However, in order to narrow down the selection it is important to know if these peptides follow all the same mode of action. In addition, the mode of action should be different from conventional antibiotics, in consequence peptide candidates can be developed further into drugs against multi-drug resistant bacteria. Here we used one short antimicrobial peptide with unknown mode of action and compared the ultrastructural changes of Escherichia coli cells after treatment with the peptide to cells treated with classic antibiotics. The key finding is that SAXS as a structure sensitive tool provides a rapid feedback on drug induced ultrastructural alterations in whole E. coli cells. We could demonstrate that ultrastructural changes depend on the used antibiotics and their specific mode of action. This is demonstrated using several well characterized antimicrobial compounds and the analysis of resulting SAXS curves by principal component analysis. To understand the result of the PCA analysis, the data is correlated with TEM images. In contrast to real space imaging techniques, SAXS allows to obtain nanoscale information averaged over approximately one million cells. The measurement takes only seconds, while conventional tests to identify a mode of action require days or weeks per single substance. The antimicrobial peptide showed a different mode of action as all tested antibiotics including polymyxin B and is therefore a good candidate for further drug development. We envision SAXS to become a useful tool within the high-throughput screening pipeline of modern drug discovery. This article is part of a Special Issue entitled: Antimicrobial peptides edited by Karl Lohner and Kai Hilpert.
多重耐药菌目前正在全球范围内破坏我们的医疗保健系统。虽然迫切需要新型抗菌药物，如抗菌肽，但确定新的作用模式既费钱又耗时，而且目前的方法无法以高通量的方式进行。在这里，我们探讨小角X射线散射（SAXS）作为一种高通量方法如何有助于对新型抗菌药物的作用模式进行分类，从而支持药物开发中的快速决策。利用天然存在的抗菌肽数据库或预测新型人工肽，可以发现许多能够杀死选定目标细菌的候选物。然而，为了缩小选择范围，了解这些肽是否遵循相同的作用模式很重要。此外，其作用模式应不同于传统抗生素，因此可以将候选肽进一步开发成抗多重耐药菌的药物。在这里，我们使用了一种作用模式未知的短抗菌肽，并将用该肽处理后的大肠杆菌细胞的超微结构变化与用经典抗生素处理的细胞进行了比较。关键发现是，SAXS作为一种结构敏感工具，能够对整个大肠杆菌细胞中药物诱导的超微结构变化提供快速反馈。我们可以证明，超微结构变化取决于所用的抗生素及其特定的作用模式。这通过使用几种特征明确的抗菌化合物并通过主成分分析对所得的SAXS曲线进行分析得到了证明。为了理解主成分分析的结果，将数据与透射电子显微镜（TEM）图像相关联。与实空间成像技术不同，SAXS能够获得大约一百万个细胞平均后的纳米级信息。测量仅需几秒钟，而确定一种作用模式的传统测试每种物质需要数天或数周时间。该抗菌肽与所有测试的抗生素（包括多粘菌素B）显示出不同的作用模式，因此是进一步药物开发的良好候选物。我们设想SAXS将成为现代药物发现高通量筛选流程中的一种有用工具。本文是由卡尔·洛纳（Karl Lohner）和凯·希尔佩特（Kai Hilpert）编辑的名为《抗菌肽》的特刊的一部分。
