Validation and Development of an Escherichia coli Riboflavin Pathway Phenotypic Screen Hit as a Small-Molecule Ligand of the Flavin Mononucleotide Riboswitch
Validation and Development of an Escherichia coli Riboflavin Pathway Phenotypic Screen Hit as a Small-Molecule Ligand of the Flavin Mononucleotide Riboswitch
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DOI：
10.1007/978-1-4939-7847-2_2
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发表时间：
2018-01-01
期刊：
影响因子：
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通讯作者：
Howe, John A.
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文献类型：
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作者：
Balibar, Carl J.;Villafania, Artjohn;Howe, John A.
核黄素生物合成途径特异性表型筛选使用的化合物库，所有具有未指明的抗生素活性，确定了一个小分子，后来命名为核黄素，其固有的抗大肠杆菌的抗菌活性被完全抑制添加外源核黄素的细菌生长培养基。核黄素抑制核黄素活性的能力，以及进一步证明核黄素抑制核黄素合成（IC 50 = 0.3 μ M），支持核黄素合成途径的组分是分子靶标。值得注意的是，抗性突变选择和全基因组测序表明，核黄素的靶点不是核黄素生物合成途径中的酶，而是核黄素单核苷酸核糖开关，这是ribB基因中编码核黄素合成关键酶的非编码结构RNA元件。尽管核糖核酸在结构上不同于天然核糖开关调节配体黄素单核苷酸肽，但核糖核酸与核糖开关的结合导致ribB表达的有效抑制以及核黄素生物合成和细菌生长的抑制。基于细胞的核糖开关调节基因报告基因测定以及体外核糖开关RNA适体结合测定，这两种测定在本文中与核黄素途径特异性筛选一起沿着详细描述，被开发以进一步验证核糖核酸的作用机制，并促进发现更有效的类似物。
A riboflavin biosynthesis pathway-specific phenotypic screen using a library of compounds, all with unspecified antibiotic activity, identified one small molecule later named ribocil, for which intrinsic antibacterial activity against Escherichia coli was completely suppressed by addition of exogenous riboflavin to the bacterial growth medium. The ability of riboflavin to suppress the activity of ribocil, and further demonstration that ribocil inhibited riboflavin synthesis (IC50 = 0.3 mu M), supported that a component of the riboflavin synthesis pathway was the molecular target. Remarkably, resistance mutation selection and whole-genome sequencing showed that the target of ribocil was not an enzyme in the riboflavin biosynthesis pathway, but instead the flavin mononucleotide riboswitch, a noncoding structural RNA element in the ribB gene that encodes a key riboflavin synthesis enzyme. Although ribocil is structurally distinct from the natural riboswitch regulatory ligand flavin mononucleotide, ribocil binding to the riboswitch results in efficient repression of ribB expression and inhibition of riboflavin biosynthesis and bacterial growth. A cell-based riboswitch regulated gene reporter assay as well as an in vitro riboswitch RNA aptamer-binding assay, both of which are described in detail here along with the riboflavin pathway-specific screen, were developed to further validate the mechanism of action of ribocil and to facilitate the discovery of more potent analogues.