Division of Biological Sciences, University of California-San Diego, La Jolla, California, USA.
Entasis Therapeutics, Gatehouse Park BioHub, Waltham, Massachusetts, USA.
J Bacteriol. 2021 Sep 8;203(19):e0010521. doi: 10.1128/JB.00105-21.
In this study, we sought to determine whether an assay for studying antibiotic mechanisms of action could provide insight into the activity of compounds that may inhibit multiple targets. Thus, we conducted an activity screen of 31 structural analogs of rhodanine-containing pan-assay interference compounds (PAINS). We identified nine active molecules against Escherichia coli and classified them according to their mechanisms of action. The mechanisms of action of PAINS are generally difficult to identify due to their promiscuity. However, we leveraged bacterial cytological profiling, a fluorescence microscopy technique, to study these complex mechanisms. Ultimately, we found that although some of our molecules promiscuously inhibit multiple cellular pathways, a few molecules specifically inhibit DNA replication despite structural similarity to related PAINS. A genetic analysis of resistant mutants revealed thymidylate kinase (essential for DNA synthesis) as an intracellular target of some of these rhodanine-containing antibiotics. This finding was supported by activity assays, as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the half-maximal inhibitory concentration and MIC displayed the greatest specificity for inhibition of the DNA replication pathway, despite containing a rhodamine moiety. Although it is thought that PAINS cannot be developed as antibiotics, this work showcases novel inhibitors of E. coli thymidylate kinase. Moreover, perhaps more importantly, this work highlights the utility of bacterial cytological profiling for studying the specificity of antibiotics and demonstrates that bacterial cytological profiling can identify multiple pathways that are inhibited by an individual molecule. We demonstrate that bacterial cytological profiling is a powerful tool for directing antibiotic discovery efforts because it can be used to determine the specificity of an antibiotic's mechanism of action. By assaying analogs of PAINS, molecules that are notoriously intractable and nonspecific, we (surprisingly) identify molecules with specific activity against E. coli thymidylate kinase. This suggests that structural modifications to PAINS can confer stronger inhibition by targeting a specific cellular pathway. While inhibition assays are susceptible to false-positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific activity.
在这项研究中，我们试图确定研究抗生素作用机制的测定方法是否可以深入了解可能抑制多个靶标的化合物的活性。因此，我们对 31 种含绕丹宁的泛分析干扰化合物（PAINS）结构类似物进行了活性筛选。我们鉴定了 9 种针对大肠杆菌的活性分子，并根据其作用机制对其进行了分类。由于 PAINS 的混杂性，其作用机制通常难以识别。然而，我们利用细菌细胞形态分析这一荧光显微镜技术来研究这些复杂的机制。最终，我们发现，尽管我们的一些分子混杂地抑制了多种细胞途径，但一些分子尽管与相关 PAINS 结构相似，但特异性地抑制 DNA 复制。对耐药突变体的遗传分析表明，胸苷酸激酶（DNA 合成所必需）是这些含绕丹宁抗生素的一些细胞内靶标。这一发现得到了活性测定的支持，以及利用胸苷酸激酶过表达系统进行的实验的支持。显示出半数最大抑制浓度和 MIC 的最大抑制 DNA 复制途径的类似物，尽管含有罗丹明部分，但显示出最大的特异性。尽管人们认为 PAINS 不能被开发成抗生素，但这项工作展示了新型大肠杆菌胸苷酸激酶抑制剂。此外，也许更重要的是，这项工作突出了细菌细胞形态分析在研究抗生素特异性方面的实用性，并表明细菌细胞形态分析可以识别单个分子抑制的多个途径。我们证明，细菌细胞形态分析是指导抗生素发现工作的有力工具，因为它可以用于确定抗生素作用机制的特异性。通过对 PAINS 的类似物进行测定，这些分子是众所周知的难以处理和非特异性的，我们（令人惊讶地）鉴定出了针对大肠杆菌胸苷酸激酶具有特异性活性的分子。这表明，通过针对特定的细胞途径进行结构修饰，可以赋予 PAINS 更强的抑制作用。虽然抑制测定容易产生假阳性结果（尤其是来自 PAINS 的结果），但细菌细胞形态分析提供了分辨率，可以识别具有特定活性的分子。
