Bacterial DnaA Initiator Protein: A Target for Novel Antibiotics

细菌 DnaA 起始蛋白：新型抗生素的靶点

• 批准号: 7393957
• 负责人: Michelle M. Butler
• 金额: $ 29.39万
• 依托单位: MICROBIOTIX, INC
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-02-01 至 2010-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7393957
• 关键词: Animal Model; Anti-Bacterial Agents; Antibiotics; Bacteria; Bacterial Infections; Binding; Biochemical; Biological Assay; Biological Factors; Cells; Chemicals; Class; Condition; DNA; DNA Binding; DNA Synthesis Inhibition; DNA biosynthesis; DNA chemical synthesis; Development; DnaB helicase; Drug Design; E coli DnaA protein; Effectiveness; Engineering; Escherichia coli; Genomics; Goals; Growth; Hydrolysis; Infection; Lead; Libraries; Mammalian Cell; Measures; Mediating; Multi-Drug Resistance; Outcome; Pathway interactions; Pharmaceutical Chemistry; Pharmaceutical Preparations; Phase; Phenotype; Protein Biosynthesis; Proteins; Protocols documentation; RNA; Rate; Replication Origin; Research; Research Design; Research Project Grants; Resistance; Screening procedure; Secondary to; Series; Signal Transduction; Specificity; Structure; Test Result; Testing; Therapeutic; TimeLine; Toxic effect; base; cytotoxicity; day; design; drug development; exhaust; high throughput screening; inhibitor/antagonist; innovation; mutant; novel; pathogen; pre-clinical; research clinical testing; resistance mechanism; small molecule

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.

描述（由申请方提供）：我们打算通过关注经过充分验证的途径中未充分开发的靶标来开发新的化学类别的抗菌药物。具体来说，我们将开发和应用高通量筛选大肠杆菌DnaA蛋白的抑制剂，这是DNA复制途径中的一个重要靶点。我们的总体目标是确定特定的DnaA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DnaA测定用于高通量筛选应用。我们将使用该测定来筛选超过100，000个离散小分子化合物和纯化天然产物的多样化文库。高通量筛选将测量E. DnaA抑制剂对大肠杆菌生长的影响。在控制条件下，E.大肠杆菌菌株不能生长，这是由于突变体DnaA对DNA合成的工程化致死过度起始。该检测试剂盒将检测多种必需DnaA功能中任一种的抑制剂，包括DnaA寡聚化、ATP结合和水解、复制起点处DNA解旋或DnaB解旋酶装载剂的募集。我们将确认命中并消除非特异性DNA结合化合物。来自该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中测试抗菌效力。将评价DNA复制的特异性和对培养物中哺乳动物细胞的细胞毒性以及生化（基于靶标）选择性，以生成一系列经验证的命中结果。本研究的具体目标是：（1）建立一种高通量筛选大肠杆菌必需DnaA蛋白的方法;（2）筛选不同的化合物库，以鉴定和确认大肠杆菌的抑制剂。coliDnaA;和（3）对光谱、效力、机制和选择性确定筛选命中的优先级。在第二阶段，我们将更详细地描述验证命中的作用机制，并利用合理的药物设计方法优化这些结构中最有前途的结构，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，并且很少有新的抗生素正在开发中。该提案描述了一种新型的筛选试验，可以识别DnaA的抑制剂，DnaA是一种以前未开发的参与DNA合成起始的蛋白质。