Bacterial DnaA Initiator Protein: A Target for Novel Antibiotics

描述（由申请人提供）：我们打算通过在经过充分验证的途径中专注于未开发的靶点来开发新的化学类抗菌药物。具体来说，我们将开发并应用高通量筛选大肠杆菌DNA蛋白抑制剂，这是DNA复制途径中的重要靶点。我们的总体目标是鉴定特定的dna抑制剂，并将其开发成新型抗生素，以治疗耐药细菌感染。在第一阶段，我们将优化基于细胞的dna检测，用于高通量筛选应用。我们将使用该试验筛选超过100,000个独立的小分子化合物和纯化的天然产物的多样化文库。高通量筛选将测量dna抑制剂对大肠杆菌生长的刺激。在控制条件下，大肠杆菌菌株无法生长，这是由于基因工程导致突变DNA合成的致死过度启动。该检测将检测任何多种基本DnaA功能的抑制剂，包括DnaA寡聚、ATP结合和水解、DNA在复制起点的解绕或DnaB解旋酶装载器的募集。我们将确认命中并消除非特异性dna结合化合物。从这一筛选中获得的有希望的化合物将在一组革兰氏阴性和革兰氏阳性细菌病原体中进行抗菌效力测试。将对培养的哺乳动物细胞的DNA复制特异性和细胞毒性以及生化（基于靶标的）选择性进行评估，以产生一系列经过验证的命中。具体目的是：(1)建立大肠杆菌必需dna蛋白的高通量筛选方法；(2)筛选多种化合物文库，鉴定和确认大肠杆菌dna抑制剂；(3)根据谱、效价、机制和选择性对已确定的筛选靶点进行排序。在第二阶段，我们将更详细地描述验证命中点的作用机制，并利用合理的药物设计方法优化最有希望的这些结构，以开发抗菌先导化合物。

DESCRIPTION (provided by applicant): We intend to develop new chemical classes of antibacterials by focusing on an under-exploited target in a well- validated pathway. Specifically, we will develop and apply a high-throughput screen for inhibitors of the Escherichia coli DnaA protein, an essential target in the DNA replication pathway. Our overall goal is to identify specific DnaA inhibitors and develop them into novel antibiotics in order to treat resistant bacterial infections. In Phase I, we will optimize a cell-based DnaA assay for a high-throughput screening application. We will use this assay to screen a diverse library of over 100,000 discrete small molecule compounds and purified natural products. The high-throughput screen will measure stimulation of E. coli growth by DnaA inhibitors. Under control conditions, the E. coli strain is unable to grow due to the engineered lethal overinitiation of DNA synthesis by mutant DnaA. This assay will detect inhibitors of any of the multiple essential DnaA functions, including DnaA oligomerization, ATP binding and hydrolysis, unwinding of DNA at the replication origin or recruitment of the DnaB helicase loader. We will confirm hits and eliminate non- specific DNA-binding compounds. Promising compounds from this screen will be tested for antibacterial potency across a panel of Gram-negative and Gram-positive bacterial pathogens. Specificity for DNA replication and cytotoxicity to mammalian cells in culture as well as biochemical (target-based) selectivity will be evaluated to generate a series of validated hits. The specific aims are to (1) develop a high-throughput screening assay for the essential DnaA protein of E. coli; (2) screen a diverse compound library to identify and confirm inhibitors of E. coli DnaA; and (3) prioritize confirmed screening hits for spectrum, potency, mechanism and selectivity. In Phase II, we will characterize the mechanism of action of the validated hits in more detail and optimize the most promising of these structures utilizing a rational drug design approach to develop antibacterial lead compounds. Multi-drug resistance is reducing the effectiveness of current antibiotics and very few new antibiotics are in the development pipeline. This proposal describes a new type of screening assay that can identify inhibitors of DnaA, a previously unexploited protein involved in the initiation of DNA synthesis.