INHIBITORS OF BACTERIAL RNA POLYMERASE: "SWITCH REGION"

描述（由申请人提供）：初步工作确定了三种天然产物-聚酮衍生的α-吡喃酮粘液吡喃酮、Dolyketide衍生的α-吡喃酮珊瑚吡喃酮和聚酮衍生的大环内酯ripostatin-通过与RNAP“开关区”相互作用抑制细菌RNA聚合酶（RNAP），RNAP“开关区”是介导RNAP结合并保留转录中的DNA模板所需的构象变化的结构元件。所述化合物不抑制真核RNAP I、RNAP II或RNAP III。所述化合物有效地抑制革兰氏阳性和革兰氏阴性细菌生长，与目前临床上用于治疗细菌感染的细菌RNAP抑制剂（利福霉素抗菌剂、利福平、利福喷丁和利福必汀）没有交叉抗性，并且与正在评估的用于未来临床上用于治疗细菌感染的其他细菌RNAP抑制剂没有交叉抗性。拟议的工作将使用X射线晶体学，系综和单分子荧光共振能量转移，单分子纳米操纵，分子克隆，替代宿主表达，基于结构的筛选和从头筛选，以解决四个具体目标： 具体目标1：RNAP与转换区靶点抑制剂复合物结构的确定具体目标2：转换区靶点抑制剂抑制RNAP的机制的确定具体目标3：转换区靶点抑制剂生物合成基因簇的克隆、表征和替代宿主表达 具体目标4：新型开关区靶点抑制剂的鉴定和表征。 这些结果将使新的广谱抗菌剂的开发成为可能，这些抗菌剂将有效地对抗对目前使用的抗菌剂具有抗性的细菌菌株。因此，研究结果将直接关系到公共卫生和制定针对可能用于生物战或生物恐怖主义的细菌菌株的对策。此外，研究结果将有助于了解国家行动方案的结构和功能，并为分析国家行动方案的结构和功能提供工具。

DESCRIPTION (provided by applicant): Preliminary work establishes that three natural products-the polyketide-derived a-pyrone myxopyronin, the Dolyketide-derived a-pyrone corallopyronin, and the polyketide-derived macrocylic lactone ripostatin-inhibit bacterial RNA polymerase (RNAP) through interactions with the RNAP "switch region," a structural element that mediates conformational changes required for RNAP to bind and retain the DNA template in transcription. The compounds do not inhibit eukaryotic RNAP I, RNAP II, or RNAP III. The compounds potently inhibit Gram-positive and Gram-negative bacterial growth, exhibit no cross-resistance with the inhibitors of bacterial RNAP in current clinical use in therapy of bacterial infection (the rifamycin antibacterial agents, rifampicin, rifapentine, and rifabutin), and exhibit no cross-resistance with other inhibitors of bacterial RNAP under evaluation for future clinical use in therapy of bacterial infection. The proposed work will use x-ray crystallography, ensemble and single-molecule fluorescence resonance energy transfer, single-molecule nanomanipulation, molecular cloning, surrogate-host expression, structure-based screening, and de novo screening, to address four specific aims: Specific Aim 1: Determination of structures of complexes of RNAP with switch-region-target inhibitors Specific Aim 2: Determination of mechanisms of inhibition of RNAP by switch-region-target inhibitors Specific Aim 3: Cloning, characterization, and surrogate-host expression of biosynthetic gene clusters for switch-region-target inhibitors Specific Aim 4: Identification and characterization of novel switch-region-target inhibitors. The results will enable development of new broad-spectrum antibacterial agents that will be effective against bacterial strains resistant to currently used antibacterial agents. As such, the results will have direct relevance to public health and to development of countermeasures against bacterial strains that could be used in biowarfare or bioterrorism. In addition, the results will contribute to understanding RNAP structure and function and will provide tools for analysis of RNAP structure and function.