Institut für Pharmazeutische Chemie, Philipps-Universität Marburg, Marburg, Germany.
Zentrum für Tumor- und Immunbiologie, Philipps-Universität Marburg, Marburg, Germany.
Antimicrob Agents Chemother. 2021 Jul 16;65(8):e0030021. doi: 10.1128/AAC.00300-21.
RNase P is an essential enzyme responsible for tRNA 5'-end maturation. In most bacteria, the enzyme is a ribonucleoprotein consisting of a catalytic RNA subunit and a small protein cofactor termed RnpA. Several studies have reported small-molecule inhibitors directed against bacterial RNase P that were identified by high-throughput screenings. Using the bacterial RNase P enzymes from Thermotoga maritima, Bacillus subtilis, and Staphylococcus aureus as model systems, we found that such compounds, including RNPA2000 (and its derivatives), iriginol hexaacetate, and purpurin, induce the formation of insoluble aggregates of RnpA rather than acting as specific inhibitors. In the case of RNPA2000, aggregation was induced by Mg ions. These findings were deduced from solubility analyses by microscopy and high-performance liquid chromatography (HPLC), RnpA-inhibitor co-pulldown experiments, detergent addition, and RnpA titrations in enzyme activity assays. Finally, we used a B. subtilis RNase P depletion strain, whose lethal phenotype could be rescued by a protein-only RNase P of plant origin, for inhibition zone analyses on agar plates. These cell-based experiments argued against RNase P-specific inhibition of bacterial growth by RNPA2000. We were also unable to confirm the previously reported nonspecific RNase activity of S. aureus RnpA itself. Our results indicate that high-throughput screenings searching for bacterial RNase P inhibitors are prone to the identification of "false positives" that are also termed an-ssay terference compound (PAINS).
RNase P 是一种负责 tRNA 5'-端成熟的必需酶。在大多数细菌中，该酶是一种由催化 RNA 亚基和一种称为 RnpA 的小蛋白辅因子组成的核糖核蛋白。有几项研究报道了针对细菌 RNase P 的小分子抑制剂，这些抑制剂是通过高通量筛选发现的。使用 Thermotoga maritima、Bacillus subtilis 和 Staphylococcus aureus 的细菌 RNase P 酶作为模型系统，我们发现这些化合物，包括 RNPA2000（及其衍生物）、iriginol 六乙酸酯和 purpurin，会诱导 RnpA 形成不溶性聚集体，而不是作为特异性抑制剂。在 RNPA2000 的情况下，聚集是由镁离子诱导的。这些发现是通过显微镜和高效液相色谱 (HPLC) 的溶解度分析、RnpA-抑制剂共沉淀实验、添加去污剂和酶活性测定中的 RnpA 滴定得出的。最后，我们使用了一种 B. subtilis RNase P 耗尽菌株，其致死表型可以被植物来源的仅蛋白质 RNase P 挽救，用于琼脂平板上的抑菌圈分析。这些基于细胞的实验表明，RNPA2000 对细菌生长没有特异性的 RNase P 抑制作用。我们也无法证实先前报道的 S. aureus RnpA 本身的非特异性 RNase 活性。我们的结果表明，高通量筛选寻找细菌 RNase P 抑制剂容易识别“假阳性”，也称为非特异性抑制化合物（PAINS）。
