A Fluorescence Displacement Assay for the Biotin Biosynthetic Enzyme BioA

描述（由申请人提供）：结核分枝杆菌是全球细菌性传染病死亡的主要原因。耐药菌株（MDR和XDR-TB）是一个新出现的问题，并且可能在美国导致重大问题，因为超过一半的病例发生在外国出生的人身上。生物素的生物合成最近被确定为开发新的抗结核药物的一个有吸引力的目标。在这个提议中，我们描述了设计，开发和优化一种新的hts -a测定方法，该方法催化了结核分枝杆菌中生物素的第二步生物合成。本申请的目的是使用我们的新型HTS法筛选BioA，然后对确定的命中进行二次和三次筛选。在第一个具体目标中，我们将为一级和二级分析准备化学和生化试剂。在第二个具体目标中，我们将使用正交LC-MS为基础的分析开发和筛选命中。在第三个特定目标中，我们将使用过表达和低表达BioA的耻垢分枝杆菌突变株进行第三次筛选，以获得生物活性并表征靶特异性。

DESCRIPTION (provided by applicant): Mycobacterium tuberculosis is the leading cause of bacterial infectious disease deaths worldwide. Drug-resistant strains (MDR and XDR-TB) are an emerging problem and could lead to significant issues in the US because over half of the cases are in foreign born persons. Biotin biosynthesis has recently been identified as an attractive target for the development of new ant tubercular agents. In this proposal we describe the design, development, and optimization of a novel HTS-assay for BioA, which catalyzes the second step of biotin biosynthesis in Mtb. The objectives of this application are to screen BioA using our novel HTS assay and then perform secondary and tertiary screening of identified hits. In the first specific aim, we will prepare chemical and biochemical reagents for the primary and secondary assays. In the second specific aim, we will develop and screen hits using an orthogonal LC-MS based assay. In the third specific aim, we will perform tertiary screening using mutant strains of M. smegmatis that over- and under-express BioA to access biological activity and characterize on-target specificity.