DNA helicase and primase inhibitors for biodefense

描述(申请人提供)：该项目的长期目标是鉴定炭疽杆菌解旋酶和底物酶的特定抑制剂，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至关重要，因为它们可能对自然和工程抗药性形式的生物恐怖分子有效，而且没有预先存在的抗药性机制。该项目采用的策略是在经过充分验证的途径中利用各种合成和天然产物化合物筛选新的靶点，以确定新的抗生素结构。关键的DNA复制途径的两个关键成分，解旋酶(Dna B)和启动酶(Dna G)，它们在复制过程中起早期作用并催化限速步骤，但尚未被探索用于发现生物防御剂。在所研究的所有细菌物种中，这两种必需的酶作为一个复合体共同作用，启动DMA复制的聚合步骤。这些靶基因将被克隆、过度表达，并证实它们在炭疽杆菌中的重要性。活性蛋白将被提纯并用于开发一种基于荧光共振能量转移(FRET)的新型解旋酶-Primase高通量Put分析方法。这一筛选将应用于100,000个离散小分子化合物和用于抑制剂的纯化天然产物的多样化资料库。这种筛选试验的新颖之处在于它能够同时检测对两个偶联反应中每一个反应的抑制，导致分析结果立即去卷积，并消除许多非特异性的打击。筛选结果将得到确认，任何剩余的非特异性DNA结合化合物将通过抑制第三复制功能(DMA聚合酶IMC)的快速二次检测来消除。化合物将被测试DNA复制的特异性，并通过它们的抑制机制来表征。最后，将测试化合物在培养中对哺乳动物细胞的毒性，以及对真核聚合酶α和解旋酶缺乏活性，从而收集经过验证的HITS。

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.