INHIBITORS OF BACTERIAL RNA POLYMERASE: "SWITCH REGION"

初步工作确定了三种天然产物-聚酮衍生的a-吡喃酮粘焦宁， 多利酮衍生的α-吡喃酮珊瑚吡喃酮和聚酮衍生的大环内酯Ripostatin抑制剂 细菌RNA聚合酶（RNAP）通过与RNAP“开关区”（一种结构元件）的相互作用 介导RNAP结合并保留DMA模板所需的构象变化， 转录。所述化合物不抑制真核RNAP I、RNAP II或RNAP III。化合物 明显抑制革兰氏阳性菌和革兰氏阴性菌的生长， 目前临床上用于治疗细菌感染的细菌RNAP的抑制剂（抗细菌的泰利福霉素 药物，利福平，利福喷丁和利福贝汀），并且与其他细菌性大肠杆菌抑制剂不表现出交叉耐药性。 RNAP正在评估未来在细菌感染治疗中的临床应用。 这项工作将使用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，以实现四个具体目标： 具体目的1：确定RNAP与转换区靶点抑制剂的复合物的结构 具体目的2：确定开关区靶点抑制剂抑制RNAP的机制 具体目标3：生物合成基因簇的克隆、表征和替代宿主表达 转换区靶点抑制剂 具体目标4：新型开关区靶点抑制剂的鉴定和表征 这些结果将有助于开发新的广谱抗菌剂， 对目前使用的抗菌剂具有抗性的细菌菌株。因此，其结果将直接 与公共卫生的相关性以及与针对可能 用于生物战或生物恐怖主义此外，研究结果将有助于理解RNAP结构 并将提供分析国家行动方案区域办事处结构和职能的工具。

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 Dacterial 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 DMAtemplate in transcription. The compounds do not inhibit eukaryotic RNAP I, RNAP II, or RNAP III. The compounds ootently inhibit Gram-positive and Gram-negative bacterial growth, exhibit no cross-resistance with the nhibitors of bacterial RNAP in current clinical use in therapy of bacterial infection (therifamycin 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 wilt 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.