在这项研究中,我们试图确定研究抗生素作用机制的体内试验是否可以深入了解可能抑制多种靶标的化合物的活性.因此,我们对含罗丹宁的泛测定干扰化合物(PAINS)的31种结构类似物进行了活性筛选。我们确定了9种针对大肠杆菌的活性分子,并根据其体内作用机制对其进行了分类。PAINS的作用机制通常由于其滥交而难以识别。然而,我们利用细菌细胞学分析,荧光显微镜技术,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多种细胞途径,一些分子特异性抑制DNA复制,尽管结构相似性相关的疼痛。对抗性突变体的遗传分析显示,胸苷酸激酶(DNA合成所必需的)是其中一些含罗丹宁的抗生素的细胞内靶标。这一发现得到了体外活性测定的支持,以及利用胸苷酸激酶过表达系统的实验。显示体外半最大抑制浓度和体内MIC的类似物对DNA复制途径的抑制表现出最大的特异性。尽管含有罗丹明部分。虽然人们认为疼痛不能作为抗生素开发,这项工作展示了大肠杆菌胸苷酸激酶的新型抑制剂。此外,也许更重要的是,这项工作强调了细菌细胞学谱分析在研究抗生素体内特异性方面的实用性,并证明细菌细胞学谱分析可以识别出被单个分子抑制的多种途径.重要性我们证明细菌细胞学分析是指导抗生素发现工作的有力工具,因为它可用于确定抗生素体内作用机制的特异性。通过测定疼痛的类似物,众所周知,难以处理和非特异性的分子,我们(令人惊讶地)鉴定了对大肠杆菌胸苷酸激酶具有特异性活性的分子。这表明对PAINS的结构修饰可以通过靶向特定的细胞途径而赋予更强的抑制作用。虽然体外抑制试验易受假阳性结果的影响(尤其是PAINS),细菌细胞学分析提供了识别具有特定体内活性的分子的分辨率。
In this study, we sought to determine whether an in vivo 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 in vivo 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 in vitro activity assays, as well as experiments utilizing a thymidylate kinase overexpression system. The analog that demonstrated the half-maximal inhibitory concentration in vitro and MIC in vivo 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 in vivo specificity of antibiotics and demonstrates that bacterial cytological profiling can identify multiple pathways that are inhibited by an individual molecule. IMPORTANCE 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 in vivo 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 in vitro inhibition assays are susceptible to false-positive results (especially from PAINS), bacterial cytological profiling provides the resolution to identify molecules with specific in vivo activity.