DNA helicase and primase inhibitors for biodefense

描述（由申请人提供）：本项目的长期目标是鉴定B的特异性抑制剂。炭疽解旋酶和引发酶，并将它们开发成用于生物防御的新型抗生素。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，因为它们可能对生物恐怖分子的天然和工程抗性形式都有效，而且没有预先存在的抗性机制。该项目采用的策略是在经过充分验证的途径中筛选具有多种合成和天然产物化合物的新靶标，以鉴定新的抗生素结构。解旋酶（dnaB）和引发酶（dnaG）是DNA复制途径中的两个关键组分，它们在复制早期起作用并催化限速步骤，但尚未被用于发现生物防御剂。在研究的所有细菌物种中，这2种必需酶作为复合物一起起作用，以启动DMA复制的聚合步骤。这些目的基因将被克隆，过表达，并在B中表达。炭疽病确诊活性蛋白将被纯化并用于开发基于荧光共振能量转移（FRET）的创新偶联解旋酶-引发酶高通量测定。该筛选将应用于> 100，000个离散小分子化合物和纯化的天然产物的多样性文库中的抑制剂。该筛选试验的新颖之处在于其能够同时检测2个偶联反应中每一个的抑制，导致试验结果立即去卷积并消除许多非特异性命中。将确认筛选命中，并通过抑制第三种复制功能（DMA聚合酶IMC）的快速二次试验消除任何剩余的非特异性DNA结合化合物。将测试化合物对DNA复制的特异性，并通过其抑制机制进行表征。最后，将测试化合物对培养物中的哺乳动物细胞缺乏毒性，以及缺乏针对真核聚合酶α和解旋酶的活性，从而产生经验证的命中的集合。

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.