Bacterial DnaA Initiator Protein: A Target for Novel Antibiotics

描述(由申请人提供)：我们打算开发新的化学类抗菌药物，重点放在一个经过充分验证的途径中未被充分开发的目标。具体地说，我们将开发和应用一种高通量筛选大肠杆菌DNAA蛋白抑制剂的方法，该蛋白是DNA复制途径中的一个重要靶点。我们的总体目标是识别特定的DNAA抑制剂，并将它们开发成新的抗生素，以治疗耐药细菌感染。在第一阶段，我们将优化基于细胞的DNAA分析，以实现高通量筛选应用。我们将使用这一方法来筛选超过100,000个离散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DNAA抑制剂对大肠杆菌生长的刺激作用。在控制条件下，由于突变的Dna A对DNA合成进行了致命的过度启动，大肠杆菌菌株无法生长。这项检测将检测多种基本DNAA功能的抑制物，包括DNAA寡聚、ATP结合和水解、复制起始处DNA的解离或DNAB解旋酶加载器的招募。我们将确认HITS并消除非特异性DNA结合化合物。来自这一筛选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上进行抗菌效力测试。将评估培养中DNA复制的特异性和对哺乳动物细胞的细胞毒性以及生化(基于靶点)的选择性，以产生一系列有效的命中。其具体目的是(1)建立一种高通量的大肠杆菌必需Dna A蛋白的筛选方法；(2)筛选一个多样化的化合物文库以鉴定和确认E.coliDna A的抑制剂；以及(3)优先对已确认的筛选命中的光谱、效力、机制和选择性进行筛选。在第二阶段，我们将更详细地描述有效HITS的作用机制，并利用合理的药物设计方法优化这些结构中最有希望的结构，以开发抗菌先导化合物。 多重耐药性正在降低现有抗生素的有效性，正在开发的新抗生素寥寥无几。这项建议描述了一种新型的筛选试验，可以识别DNAA的抑制剂，DNAA是一种以前未被开发的蛋白质，参与DNA合成的启动。

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.