DNA helicase and primase inhibitors for biodefense

项目描述（由申请人提供）：本项目的长期目标是鉴定炭疽芽孢杆菌解旋酶和引物酶的特异性抑制剂，并将其开发成新型生物防御抗生素。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，因为它们可能对天然和工程抗性形式的生物恐怖分子有效，并且因为没有预先存在的抗性机制。本项目采用的策略是通过多种合成和天然产物化合物筛选经过良好验证的途径中的新靶点，以鉴定新的抗生素结构。解旋酶（helicase, dnaB）和引物酶（primase, dnaG）是dna复制途径的两个关键组分，它们在复制过程中起作用较早，并催化了一个限速步骤，但目前尚未发现生物防御剂。在所有被研究的细菌物种中，这两种必需的酶作为一个复合体一起起作用，启动DMA复制的聚合步骤。这些靶基因将被克隆、过表达，并证实它们在炭疽芽胞杆菌中的重要性。活性蛋白将被纯化并用于开发一种基于荧光共振能量转移（FRET）的创新的偶联解旋酶-引物酶高通量分析。这一筛选将应用于一个多样化的文库，包括10万个离散的小分子化合物和纯化的天然抑制剂产品。这种筛选试验的新颖之处在于它能够同时检测两种偶联反应中的每一种的抑制作用，从而立即对试验结果进行反卷积，并消除许多非特异性命中。筛选命中将被确认，任何剩余的非特异性dna结合化合物将被快速二级分析消除，以抑制第三种复制功能（DMA聚合酶IMC）。将测试化合物对DNA复制的特异性，并确定其抑制机制。最后，将测试化合物对培养的哺乳动物细胞缺乏毒性，以及缺乏对真核聚合酶和解旋酶的活性，从而产生一系列经过验证的命中。

DESCRIPTION (provided by applicant): The long-term goal of this project is to identify specific inhibitors of B. anthracis helicase and primase and develop them into novel antibiotics for bio-defense. New antibiotics based on novel chemical scaffolds are vital to the bio-defense armory because they are likely to be effective against both natural and engineered resistant forms of bio-terrorist agents and because there are no pre-existing resistance mechanisms. The strategy employed in this project is to screen new targets in well-validated pathways with a diversity of synthetic and natural product compounds to identify new antibiotic structures. 2 key components of the essential DMA replication pathway, helicase (dnaB) and primase (dnaG), which act early and catalyze a rate-limiting step in replication, have not been explored for discovery of bio-defense agents. In all bacterial species studied, these 2 essential enzymes function together as a complex to initiate the polymerization step of DMA replication. These target genes will be cloned, over-expressed, and their essentiality in B. anthracis confirmed. Active proteins will be purified and used to develop an innovative coupled helicase-primase high through put assay based on fluorescence resonance energy transfer (FRET). This screen will be applied to a diverse library of >100,000 discrete small molecule compounds and purified natural products for inhibitors. The novel aspect of this screening assay is its ability to detect inhibition of each of the 2 coupled reactions simultaneously, resulting in immediate deconvolution of the assay results and elimination of many nonspecific hits. Screening hits will be confirmed and any remaining non-specific DNA-binding compounds eliminated by rapid secondary assays for inhibition of a third replication function (DMA polymerase IMC). Compounds will be tested for specificity for DNA replication, and characterized by their mechanism of inhibition. Finally, compounds will be tested for lack of toxicity to mammalian cells in culture, and lack of activity against eukaryotic polymerase alpha and helicase, resulting in a collection of validated hits.