INHIBITORS OF BACTERIAL RNA POLYMERASE: "SWITCH REGION"

初步工作证实，三种天然产物-聚酮衍生的α-吡喃酮粘比林， 多聚酮衍生的α-吡喃酮类珊瑚吡喃和聚酮衍生的大环内酯类药物瑞波抑素 Dacterial RNA聚合酶(RNAP)通过与RNAP“开关区”的相互作用 它介导了RNAP结合和保留DMA模板所需的构象变化 抄写。这些化合物不抑制真核细胞的RNAP I、RNAP II或RNAP III。 能显著抑制革兰氏阳性和革兰氏阴性细菌的生长，与 细菌RNAP抑制剂目前在细菌感染治疗中的应用(西红霉素抗菌药物 利福平、利福喷丁和利福平)，与其他细菌抑制剂没有交叉耐药性 RNAP正在评估未来在细菌感染治疗中的临床应用。 拟议的工作将使用x射线结晶学、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，代理宿主表达， 基于结构的筛查和从头筛查，以解决四个具体目标： 具体目标1：确定RNAP与开关区靶标抑制剂的络合物的结构 特定目标2：确定开关区靶标抑制剂抑制RNAP的机制 特定目标3：生物合成基因簇的克隆、鉴定和替代宿主表达 对于开关区-靶向抑制剂 具体目标4：新的开关区靶向抑制剂的鉴定和表征 这一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 对当前使用的抗菌剂具有抗药性的细菌菌株。因此，结果将直接 与公共卫生的相关性以及针对可能被 用于生物武器或生物恐怖主义。此外，研究结果将有助于理解RNAP的结构 和功能，并将提供分析RNAP结构和功能的工具。

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.