DEVELOPMENT OF SCREENS FOR BACILLUS ANTHRACIS TARGETS

描述(由申请人提供)：基于新型化学支架的新型抗生素对生物防御武器库至关重要，主要是因为它们将有效地对抗自然和转基因抗药性形式的生物恐怖微生物。此外，缺乏预先存在的抗性等位基因储存库可能会延长它们的使用寿命。本研究的目的是发现和开发有效对抗炭疽杆菌的新型抗生素，包括该菌的抗药性形式，用于生物防御。我们的策略是筛选DNA复制途径的新抑制剂，该途径由多个基本的、药物验证的靶点组成。在第一阶段，我们将改造非致病性炭疽杆菌斯特恩菌株，以建立一个可渗透的细胞DNA复制途径的筛选。我们将验证这一筛选，优化它以实现高吞吐量，并将其应用到包含100,000多个离散化学和天然产品结构的多样化库中。抑制DNA复制的化合物将在复制试验中得到确认，并在针对两个基本DNA复制目标--DNA聚合酶III C(PolLII C)和拓扑异构酶IV(TOPO IV)的二次筛选中进行评估。这些酶将通过从炭疽杆菌基因组DNA中分离的基因的克隆和表达来制备。此外，我们将把这些二次分析应用于围绕三个已知抑制致病但非生物恐怖分子革兰氏(+)细菌中polIIIC和topo IV的化学核心建立的重点化合物文库，以确定合适的衍生物作为炭疽杆菌抑制剂进行优化。最后，我们将测试所产生的验证命中的纯度和质量、对完整的炭疽杆菌细胞的效力、抑制DNA复制的特异性、相对于培养中的哺乳动物细胞的选择性以及其他类似药物的特性。其具体目标是：(1)利用炭疽杆菌建立可渗透细胞的高通量DNA复制途径分析；(2)构建炭疽杆菌PolIII C和Topo IV的生化二次分析方法；(3)用高通量DNA复制分析筛选多样化的化合物文库并确认所产生的HITS；以及(4)证实HITS对细菌细胞的活性、DNA合成抑制的特异性和哺乳动物细胞的细胞毒性。在第二阶段，我们将优化最有希望的化合物，以开发新的先导和候选药物，用于动物感染模型的临床前测试。

DESCRIPTION (provided by applicant): New antibiotics based on novel chemical scaffolds are vital to the biodefense armory primarily because they will be effective against both natural and engineered resistant forms of bioterrorist microbes. In addition, the absence of a reservoir of pre-existing resistance alleles may prolong their useful lifetime. The goal of this research is to discover and develop novel antibiotics effective against B. anthracis, including resistant forms of this organism, for biodefense. Our strategy is to screen for novel inhibitors of the DNA replication pathway, which consists of multiple essential, drug-validated targets. In Phase I, we will engineer the non-pathogenic B. anthracis Sterne strain to build a permeable-cell DNA replication pathway screen. We will validate this screen, optimize it for high throughput, and apply it to a diverse library of over 100,000 discrete chemical and natural product structures. Compounds that inhibit DNA replication will be confirmed in replicate assays and evaluated in secondary screens against two essential DNA replication targets -- DNA polymerase III C (pol lII C) and topoisomerase IV (topo IV). These enzymes will be prepared through the cloning and expression of genes isolated from B. anthracis genomic DNA. In addition, we will apply these secondary assays to focused compound libraries built around three chemical cores known to inhibit pol IIIC and topo IV in pathogenic, but non-bioterrorist Gram(+) bacteria in order to identify suitable derivatives for optimization as B. anthracis inhibitors. Finally, we will test the resulting validated hits for purity and mass, potency on intact B. anthracis Sterne cells, specificity for inhibition of DNA replication, selectivity with respect to mammalian cells in culture, and other drug-like properties. The specific aims are to (1) develop a permeable-cell, high-throughput DNA replication pathway assay using B. anthracis Sterne; (2) construct biochemical secondary assays for pol III C and topo IV from B. anthracis; (3) screen a diverse compound library with the high-throughput DNA replication assay and confirm resulting hits; and (4) profile confirmed hits for activity on bacterial cells, specificity for DNA synthesis inhibition, and mammalian cell cytotoxicity. In Phase II, we will optimize the most promising compounds to develop novel leads and candidate drugs for pre-clinical testing in animal models of infection.