INHIBITORS OF BACTERIAL RNA POLYMERASE: "SWITCH REGION"

描述(由申请人提供)：初步工作证实，三种天然产物-聚酮衍生的a-吡喃酮粘比罗宁、多利酮衍生的α-吡喃酮珊瑚吡喃，以及聚酮衍生的大环内酯抑制细菌RNA聚合酶(RNAP)通过与RNAP“转换区”的相互作用而产生抑制细菌RNA聚合酶(RNAP)，RNAP“转换区”是RNAP结合和保持转录中DNA模板所需的构象变化。这些化合物不抑制真核细胞的RNAP I、RNAP II或RNAP III。这些化合物有效地抑制革兰氏阳性和革兰氏阴性细菌的生长，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、利福喷丁和利福布汀)没有交叉耐药性，也没有与其他正在评估中的细菌RNAP抑制剂未来临床用于细菌感染治疗的交叉耐药性。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、基于结构的筛选和从头筛选，以解决四个具体目标： 特定目的1：确定RNAP与开关区靶标抑制剂的络合物结构2：确定开关区靶标抑制剂抑制RNAP的机制3：开关区靶标抑制剂生物合成基因簇的克隆、鉴定和替代宿主表达 具体目标4：新的开关区靶标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，这一结果将直接关系到公共卫生和制定可用于生物武器或生物恐怖主义的针对细菌菌株的对策。此外，这些结果将有助于理解RNAP的结构和功能，并将为分析RNAP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.